Cysteamine in the treatment of fibrotic disease

ABSTRACT

Fibrotic diseases are characterized by the replacement of healthy tissue with scar tissue and extracellular matrix in response to tissue damage. Here we describe the reduction of extracellular matrix (ECM) deposition, interstitial fibroblasts, interstitial volume, expression of Collagen I mRNA and protein, expression of profibrotic cytokines and macrophage infiltration by Cysteamine treatment.

BACKGROUND

1. Field of the Invention

The present embodiments relate generally to compositions and methods fortreating a patient having or being at risk for developing pathologicalfibrosis.

2. Description of the Related Art

Fibrosis can occur in the lung, liver, kidney, eye, heart, and otherorgans of the body. Fibrosis can be due to toxic or infectious injury,such as cigarette smoke to the lungs or viral hepatitis infection of theliver. The causes of some fibrotic diseases are currently unknown orpoorly understood. Fibrosis is typically considered to be anirreversible process.

One such fibrotic disease is Chronic kidney disease (CKD), also known aschronic renal disease, which affects approximately 26 million Americans.CKD is characterized by the progressive loss of renal function over aprotracted period of time (i.e., months or years). CKD leads to abuildup of fluid and waste products, which affects most body systems,including blood pressure, red blood cell production, and bone density.Complications of CKD include cardiovascular disease, anemia andpericarditis. If the progression of CKD is not halted, CKD can developinto end-Stage renal disease (ESRD), or chronic renal failure (CRF),which is a severe illness where the kidneys no longer function and thepatient requires dialysis or a kidney transplant.

The most common causes of CKD are diabetes mellitus, hypertension, andglomerulonephritis, which is characterized by inflammation of theglomeruli, or small blood vessels in the kidneys. CKD is also caused bygenetic disorders, such as Polycystic Kidney Disease (PKD),characterized by the growth of multiple cysts in the kidneys whichreduce kidney function leading to kidney failure and Nephropathiccystinosis, a lysosomal storage disorder caused by defective transportof the amino acid cystine out of lysosomes. The stored cystinecrystallizes within the lysosomes, leading to widespread tissue andorgan damage. Other causes of CKD include poisons, such as the long termuse of some over-the-counter medications and trauma.

There is no cure for CKD and left untreated it usually progresses. Thegoals of treatment are to slow disease progression, treat the underlyingcauses, treat complications of disease, and when necessary, replace lostkidney function. Current strategies for slowing progression and treatingthe underlying conditions contributing to CKD include controlling bloodglucose levels, controlling high blood pressure and eating anappropriate diet. If CKD can not be controlled and progresses to kidneyfailure, dialysis or a kidney transplant are required.

SUMMARY

The present embodiments relate to the amelioration of progressiveinterstitial fibrosis by cysteamine and/or cystamine. Severalembodiments relate to the amelioration of progressive interstitialfibrosis by modulating oxidative stress. Some embodiments relate to amethod of reducing myofibroblast accumulation and interstitialmacrophage infiltration by administering cysteamine and/or cystamine.Several embodiments relate to preventing interstitial fibrosis throughthe modulation of oxidative stress and profibrotic signaling within theinterstitium. Some embodiments relate to the administration ofcysteamine and/or cystamine to reduce oxidative stress and profibroticsignaling.

Several embodiments relate to a method of treating a fibrotic diseasecomprising administering, to a patient diagnosed with the disease, aneffective amount of cysteamine and/or cystamine product, or a saltthereof; wherein the administration of cysteamine and/or cystamineproduct, or a salt thereof, results in the amelioration of the diseasein the patient. In some embodiments, the fibrotic disease isatherosclerosis, asthma, cardiac fibrosis, organ transplant fibrosis,colloid and hypertrophic scar, muscle fibrosis, pancreatic fibrosis,bone-marrow fibrosis, liver fibrosis, cirrhosis of liver andgallbladder, scleroderma, pulmonary fibrosis, diffuse parenchymal lungdisease, idiopathic interstitial fibrosis, interstitial pneumonitis,desquamative interstitial pneumonia, respiratory bronchiolitis,interstitial lung disease, acute interstitial pneumonitis, nonspecificinterstitial pneumonia, cryptogenic organizing pneumonia, lymphocyticinterstitial pneumonia, renal fibrosis, or chronic kidney disease.

Several embodiments relate to a method for treating a disorderassociated with elevated levels of interstitial extracellular matrix(ECM) in an organ, said method comprising administering, to a patientdiagnosed with the disorder, an effective amount of cysteamine and/orcystamine product, or a salt thereof; wherein the administration ofcysteamine and/or cystamine product, or a salt thereof, results in thelowering of interstitial ECM in the organ of the patient.

Some embodiments relate to the attenuation of extracellular matrixsynthesis during chronic kidney injury by cysteamine and/or cystamineproduct.

Several embodiments relate to methods of suppressing interstitial renalfibrosis by reducing the synthesis of extracellular matrix duringchronic kidney injury by administering an effective amount of cysteamineand/or cystamine product. In some embodiments, an effective amount maybe about 0.5 mg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg/25mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150mg/kg, 175 mg/kg, 200 mg/kg, and may increase by 25 mg/kg increments upto 1000 mg/kg BW cysteamine. In some embodiments, an effective amountcysteamine and/or cystamine is a total daily dose of from approximately0.25 g/m² to 4.0 g/m² body surface area. In some embodiments, aneffective amount cysteamine is at least about a total daily dose of 0.5,0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or2 g/m², or up to about 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, 2.0, 2.2, 2.5, 2.7, 3.0, or 3.5 g/m².

Several embodiments relate to preventing interstitial fibrosis throughthe modulation of oxidative stress and profibrotic signaling within theinterstitium during chronic kidney injury.

Several embodiments described herein relate to compositions and methodsfor delaying, slowing, or halting the progression of chronic kidneydisease. Some embodiments relate to a method of delaying, slowing, orhalting the progression of chronic kidney disease by cysteaminemodulation of extracellular matrix accumulation.

Several embodiments described herein relate to a method for delaying,slowing or halting the progression of chronic kidney disease from Stage1 to Stage 2, Stage 2 to Stage 3, Stage 3 to Stage 4, or Stage 4 toStage 5 by administration of an effective amount of cysteamine and/orcystamine product. In some embodiments, an effective amount may be about0.5 mg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg/25 mg/kg, 30mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 70mg/kg, 75 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg,175 mg/kg, 200 mg/kg, and may increase by 25 mg/kg increments up to 1000mg/kg BW cysteamine and/or cystamine. In some embodiments, an effectiveamount cysteamine and/or cystamine is a total daily dose of fromapproximately 0.25 g/m² to 4.0 g/m² body surface area. In someembodiments, an effective amount cysteamine and/or cystamine is at leastabout a total daily dose of 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 g/m², or up to about 0.8, 0.9, 1.0,1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.2, 2.5, 2.7, 3.0, or3.5 g/m².

Several embodiments relate to a method for treating renal fibrosis orchronic kidney disease comprising administering to a subject in needthereof a composition comprising a cysteamine product, optionallycysteamine or cystamine or a pharmaceutically acceptable salt thereof.In some embodiments, the chronic kidney disease is characterized byrenal fibrosis, glomerulosclerosis or tubulointerstitial fibrosis, or acombination thereof. In some embodiments, the method comprisespreventing chronic kidney disease, optionally wherein the subject issuffering from chronic renal insufficiency (CRI). In some embodiments,the method comprises treating a subject suffering from Stage I, II, III,IV or V chronic kidney disease. In some embodiments, the subject issuffering from nephropathy, glomerulosclerosis, glomerulonephritis,diabetes, fibrocystic kidney disease, fibrotic kidney cancer, and renalinterstitial fibrosis. In some embodiments, the composition reducesextracellular matrix deposition in the kidney. In some embodiments, thecomposition reduces the level of one or more of collagen I, collagen II,collagen IV, procollagen I, procollagen or fibronectin. In someembodiments, the composition reduces myofibroblast infiltration and/orinterstitial macrophage infiltration in the kidney. In some embodiments,the composition reduces fibrosis in the kidney. In some embodiments, thecomposition is administered less than four times/day, optionally, one,two, or three times per day. In some embodiments, the composition isadministered at a dose from 0.01 mg to 1000 mg/kg per day. In someembodiments, the composition is administered at a dose from 0.25 g/m2 to4.0 g/m2 per day. In some embodiments, the composition further comprisesa pharmaceutically acceptable carrier; excipient, or diluent. In someembodiments, the composition is a sterile pharmaceutical composition. Insome embodiments, the composition is administered for a period of atleast 3 weeks. In some embodiments, the composition is administered fora period of at least 4 weeks, 6 weeks, 8 weeks, 12 weeks, 16 weeks, 20weeks, or 24 weeks. In some embodiments, the composition is a delayed orcontrolled release dosage form that provides increased delivery of thecysteamine product to the small intestine. In some embodiments, thedelayed or controlled release dosage form comprises an enteric coatingthat releases the cysteamine composition when the composition reachesthe small intestine or a region of the gastrointestinal tract of asubject in which the pH is greater than about pH 4.5. In someembodiments, the composition is administered orally. In someembodiments, the composition is administered parenterally. In someembodiments, the composition is administered with a second agent usefulto treat renal fibrosis, chronic kidney disease, or the associateddisease state, optionally diabetes.

Several embodiments relate to a composition for use in the treatment ofrenal fibrosis or chronic kidney disease comprising a cysteamineproduct, optionally cysteamine or cystamine or a pharmaceuticallyacceptable salt thereof. In some embodiments, the composition furthercomprises a pharmaceutically acceptable carrier, excipient, or diluent.In some embodiments, the composition is a sterile pharmaceuticalcomposition. In some embodiments, the composition is a delayed orcontrolled release dosage form that provides increased delivery of thecysteamine product to the small intestine. In some embodiments, thedelayed or controlled release dosage form comprises an enteric coatingthat releases the cysteamine composition when the composition reachesthe small intestine or a region of the gastrointestinal tract of asubject in which the pH is greater than about pH 4.5. In someembodiments, the composition comprises a second agent useful to treatrenal fibrosis, chronic kidney disease, or the associated disease state,optionally diabetes.

Some embodiments relate to a method of treating CKD comprisingadministering an effective amount of cysteamine to a patient in needthereof. In some embodiments, an effective amount may be about 0.5mg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg/25 mg/kg, 30mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 70mg/kg, 75 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg,175 mg/kg, 200 mg/kg, and may increase by 25 mg/kg increments up to 1000mg/kg BW cysteamine. In some embodiments, an effective amount cysteamineis a total daily dose of from approximately 0.25 g/m² to 4.0 g/m² bodysurface area. In some embodiments, an effective amount cysteamine is atleast about a total daily dose of 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1,1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 g/m², or up to about 0.8,0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.2, 2.5,2.7, 3.0, or 3.5 g/m².

Some embodiments relate to a method of treating CKD comprisingadministering an effective amount of cystamine to a patient in needthereof. In some embodiments, an effective amount may be about 0.5mg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg/25 mg/kg, 30mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 70mg/kg, 75 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg,175 mg/kg, 200 mg/kg, and may increase by 25 mg/kg increments up to 1000mg/kg BW cystamine. In some embodiments, an effective amount cystamineis a total daily dose of from approximately 0.25 g/m² to 4.0 g/m² bodysurface area. In some embodiments, an effective amount cystamine is atleast about a total daily dose of 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1,1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 g/m², or up to about 0.8,0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.2, 2.5,2.7, 3.0, or 3.5 g/m².

Some embodiments relate to a method of treating interstitial fibrosiscomprising administering an effective amount of cysteamine to a patientin need thereof. In some embodiments, an effective amount may be about0.5 mg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg/25 mg/kg, 30mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 70mg/kg, 75 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg,175 mg/kg, 200 mg/kg, and may increase by 25 mg/kg increments up to 1000mg/kg BW cysteamine. In some embodiments, an effective amount cysteamineis a total daily dose of from approximately 0.25 g/m² to 4.0 g/m² bodysurface area. In some embodiments, an effective amount cysteamine is atleast about a total daily dose of 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1,1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 g/m², or up to about 0.8,0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.2, 2.5,2.7, 3.0, or 3.5 g/m².

Some embodiments relate to a method of treating interstitial fibrosiscomprising administering an effective amount of cystamine to a patientin need thereof. In some embodiments, an effective amount may be about0.5 mg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg/25 mg/kg, 30mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 70mg/kg, 75 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg,175 mg/kg, 200 mg/kg, and may increase by 25 mg/kg increments up to 1000mg/kg BW cystamine. In some embodiments, an effective amount cystamineis a total daily dose of from approximately 0.25 g/m² to 4.0 g/m² bodysurface area. In some embodiments, an effective amount cystamine is atleast about a total daily dose of 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1,1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 g/m², or up to about 0.8,0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.2, 2.5,2.7, 3.0, or 3.5 g/m².

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows an illustration depicting key steps in kidney scarformation. FIG. 1B shows a diagram depicting an overview of the keyparticipants in the pathogenesis of tubulo-interstitial fibrosis. FIG.1C shows a photomicrograph of renal interstitial fibrosis. FIG. 1D showsa graph depicting the correlation between renal function as measured byinulin clearance and interstitial disease score.

FIG. 2 depicts a diagram illustrating a putative model of the in vivobiology of cysteamine.

FIG. 3A shows an illustration of a kidney subject to unilateral ureteralobstruction (UUO) (left) and a normal kidney (right). FIG. 3B shows aphotomicrograph of interstitial collagen in a mouse kidney subjected tosham surgery (top panel) and UUO (bottom panel).

FIG. 4 shows a graph depicting total kidney collagen as measured byhydroxyproline concentration at day 14 after UUO in groups of mice(n=2/group) receiving intraperitoneal injections of PBS (control), 100mg/kg cysteamine HCL, 200 mg/kg cysteamine HCL or 400 mg/kg cysteamineHCL.

FIG. 5 shows a graph depicting total kidney collagen as measured byhydroxyproline concentration in individual mice receiving placebo, 200mg/kg, 400 mg/kg or 600 mg/kg cysteamine bitartrate 1 added to thedrinking water for 14 days after UUO.

FIG. 6 shows a graph depicting total kidney collagen as measured byhydroxyproline concentration at 0, 3, 7, 14, and 21 days after UUO indosage groups of mice receiving placebo, 400 mg/kg or 600 mg/kgcysteamine bitartrate.

FIG. 7A shows a graph depicting the expression ratio of Fibronectin,Procollagen I and Procollagen III in kidneys of 400 mg/kg cysteaminebitartrate treated mice 3, 7 and 14 days after UUO. FIG. 7B shows agraph depicting the expression ratio of Fibronectin, Procollagen I andProcollagen III in kidneys of 600 mg/kg cysteamine bitartrate treatedmice 3, 7 and 14 days after UUO.

FIG. 8A shows a graph depicting α-SMA interstitial staining area 7 and14 days after UUO in untreated mice and mice treated with 400 mg/kg or600 mg/kg cysteamine bitartrate. FIG. 8B shows an α-SMAimmunohistochemical photomicrograph (400×) of a kidney of an untreatedmouse 14 days after UUO. FIG. 8C shows an α-SMA immunohistochemicalphotomicrograph (400×) of a kidney 14 days after UUO of a mousereceiving 400 mg/kg cysteamine bitartrate. FIG. 8D shows an α-SMAimmunohistochemical photomicrograph (400×) of a kidney 14 days after UUOof a mouse receiving 600 mg/kg cysteamine bitartrate.

FIG. 9A shows a graph depicting F4/80-positive interstitial area 7 and14 days after UUO in untreated mice and mice treated with 400 mg/kg or600 mg/kg cysteamine bitartrate (n=4-8/group, †P<0.01). FIG. 9B shows arepresentative confocal (400×) image of F4/80-positive macrophages in akidney of an untreated mouse 14 days after UUO. FIG. 9C shows arepresentative confocal (400×) image of F4/80-positive macrophages in akidney 14 days after UUO of a mouse receiving 400 mg/kg cysteaminebitartrate. FIG. 9D shows a representative confocal (400×) image ofF4/80-positive macrophages in a kidney 14 days after UUO of a mousereceiving 600 mg/kg cysteamine bitartrate.

FIG. 10A shows a graph depicting relative mRNA transcription of TGF-βand TGF-β receptor 1 genes in 400 mg/kg cysteamine bitartrate-treatedand control mice 7 and 14 days after UUO. FIG. 10B shows a graphdepicting relative mRNA transcription of TGF-β and TGF-β receptor 1genes in 600 mg/kg cysteamine bitartrate-treated and control mice 7 and14 days after UUO.

FIG. 11 shows a graph depicting thiol content (nM/μg protein) of tissuefrom contralateral and UUO kidneys harvested at days 7, 14, and 21 frommice administered placebo or 600 mg/kg cysteamine bitartrate.

FIG. 12A depicts the metabolic pathway through which pantethine isconverted to vitamin B5 and cysteamine. FIG. 12B shows a graph depictingtotal kidney collagen as measured by hydroxyproline concentration inVanin+/+ and Vanin−/− control mice and Vanin+/+ and Vanin−/− mice 14 and21 days after UUO.

FIG. 13A shows a representative confocal image of F4/80+ interstitialmacrophages in cystinotic (Ctns−/−) mouse kidneys at 3 months. FIG. 13Bshows a representative confocal image of F4/80+ interstitial macrophagesin cystinotic (Ctns−/−) mouse kidneys at 12 months. (g=glomeruli.) FIG.13C shows a graph depicting total collagen in control (Ctns+/+)(n=8) andCtns−/− (n=4) kidneys at 3 and 12 months of age.

FIG. 14A shows a graph depicting total collagen in kidneys(contralateral and UUO) of Ctns+/+ (n=7) and Ctns−/− (n=7) mice at day14 after UUO. FIG. 14B shows a graph summarizing the quantification ofF4/80+ macrophages in comparable areas of Ctns+/+ (n=8) and Ctns−/−(n=4) day 14 UUO kidneys. FIG. 14C shows a representative confocal imageof F4/80+ interstitial macrophages in a Ctns+/+ day 14 UUO kidney. FIG.14D shows a representative confocal image of F4/80+ interstitialmacrophages in a Ctns−/− day 14 UUO kidney.

FIG. 15A shows a graph depicting the relative expression ratio ofcystinosin (Ctns) in normal (contralateral) kidney and UUO kidneys atdays 3, 7 and 14 (n=4/group). FIG. 15B shows a graph depicting therelative expression ratio of Ctns in thioglycollate peritonealmacrophages (Mphi) and in Mphi co-cultured with apoptotic renal tubularcells (+IRR MCT).

FIG. 16A shows graphs depicting the relative expression ratio of TNF-α,TNF-α Receptor, TGF-β, and TGF-β Receptor in Ctns+/+ and Ctns−/−macrophages and in Ctns+/+ and Ctns−/− macrophages after efferocytosis.FIG. 16B shows graphs depicting the relative expression levels of TNF-α,TNF-α Receptor, TGF-β, and TGF-β Receptor in Ctns+/+ and Ctns−/− UUOkidneys at day 14. († P<0.01, NS=not significant, n=6/group.)

FIG. 17 shows a graph depicting plasma cysteamine levels in high dose(600 mg/kg) cysteamine bitartrate treated mice.

FIG. 18A shows Transglutaminase 2 (TGase2) and β-actin proteinexpression in mouse liver, normal kidney and UUO kidneys at days 3, 7and 14. FIG. 18B shows a graph depicting TGase2 protein levelsnormalized to β-actin. FIG. 18C shows TGase2 and β-actin proteinexpression in UUO kidneys of untreated mice and mice treated with 400mg/kg or 600 mg/kg Cystagon®.

DETAILED DESCRIPTION

Several embodiments described herein relate to the treatment offibrosis. Fibrosis is a pathologic process, which occurs when the body'snatural healing process goes awry, leading to over production ofextracellular matrix (ECM) and scar formation in response to tissuedamage. Fibrosis formation involves the interaction between many celltypes and cytokines, and when the balance becomes profibrotic, there isfibrosis formation. There are many fibrotic diseases, including, but notlimited to, atherosclerosis, asthma, cirrhosis, scleroderma, andpulmonary fibrosis. In some embodiments, the fibrosis is fibrosis of thelung, heart, blood vessel, liver, gallbladder, kidney, skin, lung,muscle, pancreas, eye, adrenal gland, thyroid, or other organs of thebody.

Several embodiments relate to chronic kidney disease (CKD). CKD beginswith renal injury; the progression thereafter depends upon a number ofgenetic and environmental factors. Periods of injury/inflammation arefollowed by repair processes, which may result in regeneration of renalstructures and recovery of function, or may result in replacement ofrenal structures by nonfunctional matrices. Regardless of the cause, theunderlying mechanism in the progression of chronic kidney disease tokidney failure is the accumulation of scar tissue in the kidney due tofibrosis, the formation of excess fibrous connective tissue. Although itis not known what triggers fibrosis as opposed to functional repair,renal fibrosis is characterized by the loss of renal tubules andperitubular capillaries, inflammation (macrophage infiltration),accumulation of extracellular matrix proteins and the presence ofmyofibroblasts in the interstitial space. A depiction of key steps inkidney scar formation is shown in FIGS. 1A and 1B. The area occupied bythe tubules declines as the interstitial area increases. Tubular lossexplains the close relationship between interstitial fibrosis anddeclining renal function. As shown in FIG. 1D, reduced renal function asmeasured by inulin clearance is tightly correlated with interstitialdisease score.

The cellular events of renal fibrosis occur simultaneously, and often ina mutually stimulating manner. These events include increased matrixproduction, inhibition of matrix degradation, modulation of matrixreceptors to facilitate cell-matrix interactions, release of fibrogenicfactors, fibroblast activation, interstitial myofibroblast recruitment,tubular epithelial-to-mesenchymal transition, monocytic and lymphocyticcell infiltration, and cell apoptosis. The result is the replacement ofnormal structures with accumulated extracellular matrix (ECM). A summaryof matrix proteins that accumulate in the interstitium during renalfibrosis is shown at Table 1.

TABLE 1 Matrix proteins that accumulate in the interstitium during renalfibrosis Interstitial matrix proteins Collagens I, III, V, VII, XVFibronectin Tenascin Basement membrane proteins Collagen IV LamininExtracellular proteoglycans Large chondroitin sulfate proteoglycans(aggrecan, versican) Small proteoglycans (decorin, fibromodulin,biglycan) Basement membrane proteoglycans (heparin sulfate proteoglycan,perlecan) Polysaccharides and glycoproteins Hyaluronan ThrombospondinSecreted protein, acidic, and rich in cysteine (SPARC)

CKD is classified into five stages of increasing severity. Stage 1 ischaracterized by slightly diminished function, kidney damage with normalor relatively high glomerular filtration rate (GFR) (≧90 mL/min/1.73m²). Stage 2 is characterized by a mild reduction in GFR (60-89ml/min/1.73 m²) with kidney damage. Kidney damage is defined aspathological abnormalities or markers of damage, including abnormalitiesin blood or urine tests or imaging studies. Stage 3 is characterized bya moderate reduction in GFR (30-59 ml/min/1.73 m²). Stage 4 ischaracterized by severe reduction in GFR (15-29 ml/min/1.73 m²). Stage5, which is also known as established kidney failure, is characterizedby GFR <15 mL/min/1.73 m² and permanent renal replacement therapy (RRT)is required. Patients with chronic kidney disease stages 1-3 aregenerally asymptomatic, while clinical manifestations typically appearin stages 4-5. The goal of therapy is to slow down or halt theprogression of CKD to Stage 5.

Reduction in renal function is correlated with the severity oftubulointerstitial fibrosis as shown in FIG. 1C. Accordingly, slowing orstopping ECM build-up and the loss of normal kidney structuresassociated with interstitial renal fibrosis is likely to slow or haltthe progression of CKD. However, few therapeutic options exist to slowor halt the relentless expansion of interstitial extracellular matrix(ECM) leading to nephron loss and progressive decline of kidneyfunction. Described herein are the therapeutic effects of cysteamine onameliorating interstitial fibrosis and the progression of CKD.

Cysteamine plays a role in the generation of the protein glutathione(GSH), and is currently FDA approved for use in the treatment ofcystinosis, an intra-lysosomal cystine storage disorder. Cystinosis is arare inherited disorder caused by the inability to metabolize the aminoacid cystine, which accumulates as cystine crystals throughout the body.These crystals cause tissue damage, particularly in the kidney. Incystinosis, cysteamine acts by converting cystine to cysteine andcysteine-cysteamine mixed disulfide which are then both able to leavethe lysosome through the cysteine and lysine transporters respectively(Gahl et al., N Engl J Med 2002; 347(2):111-21). See FIG. 2. Within thecytosol the mixed disulfide can be reduced by its reaction withglutathione and the cysteine released can be used for further GSHsynthesis. The synthesis of GSH from cysteine is catalyzed by twoenzymes, gamma-glutamylcysteine synthetase and GSH synthetase. Thispathway occurs in almost all cell types, with the liver being the majorproducer and exporter of GSH. The reduced cysteine-cysteamine mixeddisulfide will also release cysteamine, which, in theory is then able tore-enter the lysosome, bind more cystine and repeat the process (Dohilet al., J Pediatr 2006; 148(6):764-9). In a recent study in childrenwith cystinosis, enteral administration of cysteamine resulted inincreased plasma cysteamine levels, which subsequently caused prolongedefficacy in the lowering of leukocyte cystine levels (Dohil et al., JPediatr 2006; 148(6):764-9). This may have been due to “re-cycling” ofcysteamine when adequate amounts of drug reached the lysosome. Ifcysteamine acts in this fashion, then GSH production may also besignificantly enhanced.

Cysteamine is a potent gastric acid-secretagogue that has been used inlaboratory animals to induce duodenal ulceration; studies in humans andanimals have shown that cysteamine-induced gastric acid hypersecretionis most likely mediated through hypergastrinemia. In previous studiesperformed in children with cystinosis who suffered regular uppergastrointestinal symptoms, a single oral dose of cysteamine (11-23mg/kg) was shown to cause hypergastrinemia and a 2 to 3-fold rise ingastric acid-hypersecretion, and a 50% rise in serum gastrin levels.Symptoms suffered by these individuals included abdominal pain,heartburn, nausea, vomiting, and anorexia. U.S. patent application Ser.No. 11/990,869 and published International Publication No. WO2007/089670, both claiming priority to U.S. Provisional PatentApplication No. 60/762,715, filed Jan. 26, 2006, (all of which areincorporated by reference herein in their entirety) showed thatcysteamine induced hypergastrinemia arises, in part, as a local effecton the gastric antral-predominant G-cells in susceptible individuals.The data also suggest that this is also a systemic effect of gastrinrelease by cysteamine.

Subjects with cystinosis are required to ingest oral bitartrate salt ofcysteamine (known commercially as CYSTAGON®) every 6 hours day andnight. When taken regularly, cysteamine can deplete intracellularcystine by up to 90% (as measured in circulating white blood cells), andthis had been shown to reduce the rate of progression to kidneyfailure/transplantation and also to obviate the need for thyroidreplacement therapy. Because of the difficulty in taking CYSTAGON®,reducing the required dosing improves the adherence to therapeuticregimen. International Publication No. WO 2007/089670 demonstrates thatdelivery of cysteamine to the small intestine reduces gastric distressand ulceration, increases Cmax and increases area under the curve (AUC).Delivery of cysteamine into the small intestine is useful due toimproved absorption rates from the small intestine, and/or lesscysteamine undergoing hepatic first pass elimination when absorbedthrough the small intestine. A decrease in leukocyte cystine wasobserved within an hour of treatment.

Cysteamine has other important metabolic effects that may providerenoprotective properties. For example, cysteamine reduces functions asan antioxidant as a biological thiol, which may impact fibroticpathways.

The effect of cysteamine on the degree of renal fibrosis wasinvestigated in a model of experimental unilateral ureteral obstruction(UUO). UUO is a well-characterized experimental model of renal injury,leading to tubulointerstitial fibrosis, which is a common characteristicof many chronic nephropathies. Markers of fibrosis, such as ECMdeposition, interstitial fibroblasts, interstitial volume, mRNA andprotein expression for collagen I, and macrophage infiltration are allincreased in the kidneys of UUO animals, making the UUO model a goodexperimental system for studying fibrotic diseases. FIG. 3B shows anincrease in interstitial collagen in kidneys subjected to UUO.

The effect of cysteamine on the degree of renal fibrosis in UUO wasinvestigated using two doses of cysteamine bitartrate, 400 mg/kg/day and600 mg/kg/day and compared to mice that received vehicle alone(n=8/timepoint). Cysteamine bitartrate, the bitartrate salt ofcysteamine is known commercially as Cystagon®. In these investigationsof cysteamine's effect on fibrosis, mice were subjected to UUO on Day 0and the kidneys were removed for histological assessment of fibrosis atdays 3, 7, 14 and 21. Both doses were well-tolerated and measured serumlevels of cysteamine were appropriate. Using loss of the renal tubularcell adherens junction protein E-cadherin as a marker of the degree oftubular damage, immunoblotting studies demonstrated that E-cadherinlevels were increased 1.3 fold in the cysteamine bitartrate-treated UUOmice. Further, total kidney collagen content, as a measure of fibrosisseverity, was significantly reduced by 21% in both the 400 mg/kg and 600mg/kg doses in cysteamine-treated mice at day 14. See FIG. 6. ECM genetranscription levels were significantly down-regulated in UUO kidneys ofcysteamine-treated mice: procollagen I mRNA levels were 56% lower in themice treated with 600 mg/kg at day 14; and at day 7, despite nodifference in total collagen, there was a nearly 40% reduction in kidneyfibronectin and procollagen III mRNA levels in mice treated with 400mg/kg and a nearly 60% reduction in fibronectin, procollagen I andprocollagen III at higher doses of cysteamine (600 mg/kg). See FIG. 7.Thus cysteamine treatment reduces fibrosis severity in part by loweringthe expression of ECM components after renal injury.

Myofibroblasts are the primary interstitial cells that produceextracellular matrix during chronic kidney injury as shown in FIG. 8.Myofibroblasts can be distinguished by expression of alpha-smooth muscleactin (SMA). There was a significant reduction in alpha-SMA positivemyofibroblasts by nearly 30% in both doses at day 14 incysteamine-treated mice. See FIG. 9. In addition, there was asignificant reduction in interstitial macrophage infiltration by 34% inmice treated with 600 mg/kg/day. See FIG. 9. These data show thatcysteamine bitartrate affects both myofibroblast accumulation andinterstitial macrophage infiltration.

Renal failure is accompanied by oxidative stress, which is caused byenhanced production of reactive oxygen species and impaired antioxidantdefense. Oxidative stress enhances macrophage recruitment into vascularand renal lesions by increasing the responsiveness of macrophages tochemoattractants. Cysteamine, which can act as a biological antioxidantdue to its thiol converting properties, may provide renal protectiveeffects that can be attributed in part to its role as an antioxidant.Total kidney thiol content, a measure of antioxidant status, wassignificantly increased by 36% in high dose cysteamine-treated mice (600mg/kg) compared to controls, indicating that cysteamine modulatesantioxidant status at early time points. These data show that cysteamineaffects both myofibroblast accumulation and interstitial macrophageinfiltration in association with reduced oxidative stress within theinterstitium during chronic kidney injury.

Expression of profibrotic cytokines, such as transforming growth factorβ (TGF-β) and its receptor (TGF-(β R1) are increased in renal fibrosis.Further, activation of TGF-β signaling induces renal fibrosis leading toend Stage kidney disease. As shown in FIG. 10 and Table 2, cysteaminetreatment decreases the expression of profibrotic cytokines in UUOkidneys. Accordingly, cysteamine asserts its renal-protective affects,in part, by inhibiting the expression of the profibrotic cytokines,TGF-β, and TGF-β R1.

Cysteamine is endogenously produced by the hydrolosis of pantathine intocysteamine and pantothenic acid (vitamin B₅). See FIG. 12A. Vanin-1 isan epithelial enzyme with pantetheinase activity which catalyzes themetabolism of pantathine. Vanin-1 is highly expressed in the kidney.Mice lacking Vanin-1 have decreased/absent cysteamine levels inepithelial cells. As shown in FIG. 12B, studies on vanin−/− micedemonstrated that there was a non-statistically significant, increase intotal collagen compared to littermate control mice (P=0.2) at day 21after UUO.

Transglutaminases (TGases) are a family of enzymes that catalyze thetransamidation reaction between the γ-carboxyamide of a peptide-boundglutamine residue and the ε-amino group of a peptide-bound lysineresidue or the primary amine group of a polyamine via forming athioester acylenzyme intermediate at the active site cysteine. Thus,TGase activity produces cross-linked proteins or amine conjugates. Onetype of TGase, TGase2, acts to cross link ECM proteins, particularlyCollagen III. Cross linking renders proteins resistant to degradation,which is thought to result in accelerated matrix deposition. As shown inFIG. 18A, TGase2 is expressed both in the normal and in the UUO kidney.Cystamine (β,β′-diaminodiethyl disulfide), a thiolamine joined by adisulfide bridge between two cysteamines (β-mercaptoethylamine), can actas a TGase2 inhibitor. It has been reported that cystamine may act as aTGase inhibitor due to the presence of the of the disulfide bond (Jeonet al., Exp. Mol. Med. 2004; 36(6):576-81). Cysteamine is the reducedform of cystamine, and has been speculated to have anti-fibroticactivity as an inhibitor of TGase2. Cysteamine, however, has been shownto be a less potent inhibitor of TGase than both cystamine and primaryamines in vitro (Jeon et al., Exp. Mol. Med. 2004; 36(6):576-81).Additionally, no difference in TGase2 protein expression is observed inUUO kidneys treated with 400 mg/kg or 600 mg/kg cysteamine. See FIG.18C. Thus, inhibition of matrix cross-linking by inhibition of TGase2 isunlikely to be a major factor in the protection of renal function bycysteamine, and the role of cysteamine in inhibiting TGase2 in fibrosisremains unclear.

As shown herein, cysteamine treatment studies establish cysteamine'ssignificant anti-fibrotic effects. Cysteamine treatment decreases thetranscription of ECM genes in response to tissue damage. Cysteaminetreatment reduces interstitial collagen deposition in models of fibrosisand decreases myofibroblast and macrophage accumulation. Accordingly,cysteamine treatment reduces the severity of fibrosis associated withtissue damage. There is no evidence, however, that cysteamine assertsits anti-fibrotic effects through TGase2 modulation.

Reduction of fibrosis by administration of cysteamine presents atreatment for a wide range of fibrotic diseases. Several embodimentsdescribed herein relate to a pharmaceutical composition comprisingcysteamine or any pharmaceutically acceptable salts, analogs,derivatives, conjugates, and metabolites thereof. Several embodimentsdescribed herein relate to a pharmaceutical composition comprisingprodrugs of cysteamine that can, for example, be readily metabolized inthe body to produce cysteamine.

As used herein, the term “patient” refers to the recipient of atherapeutic treatment and includes all organisms within the kingdomanimalia. In preferred embodiments, the animal is within the family ofmammals, such as humans, bovine, ovine, porcine, feline, buffalo,canine, goat, equine, donkey, deer, and primates. The most preferredanimal is human.

As used herein, the term “treat” or any variation thereof (e.g.,treatment, treating, etc.), refers to any treatment of a patientdiagnosed with a biological condition, such as renal interstitialfibrosis, atherosclerosis, asthma, cardiac fibrosis, organ transplantfibrosis, colloid and hypertrophic scar, bone-marrow fibrosis, liverfibrosis, cirrhosis of liver and gallbladder, scleroderma, pulmonaryfibrosis, Diffuse parenchymal lung disease, idiopathic interstitialfibrosis, interstitial pneumonitis, desquamative interstitial pneumonia,respiratory bronchiolitis interstitial lung disease, acute interstitialpneumonitis, nonspecific interstitial pneumonia, cryptogenic organizingpneumonia, lymphocytic interstitial pneumonia, renal fibrosis, and/orchronic kidney disease, using the materials and/or methods of theinvention. The term treat, as used herein, includes: (i) preventing ordelaying the presentation of symptoms associated with the biologicalcondition of interest in an at-risk patient who has yet to displaysymptoms associated with the biological condition (e.g. preventing thepresentation of symptoms in a patient who is suffering from chronickidney disease stages 1-3, preventing organ transplant fibrosis, etc.);(ii) ameliorating the symptoms associated with the biological conditionof interest in a patient diagnosed with the biological condition (e.g.,fluid accumulation in a patient suffering from chronic kidney disease);(iii) preventing, delaying, or ameliorating the presentation of symptomsassociated with complications, conditions, or diseases associated withthe biological condition of interest (e.g., preventing, delaying orameliorating the presentation of cardiovascular disease, anemia,hypertension and/or renal osteodystrophy) in either an at-risk patientor a patient diagnosed with the biological condition; (iv) slowing,delaying or halting the progression of the biological condition (e.g.,slowing, delaying or halting the progression of chronic kidney diseasefrom Stage 1 to Stage 2, Stage 2 to Stage 3, Stage 3 to Stage 4, orStage 4 to Stage 5; delaying, slowing or halting the progression ofliver fibrosis to cirrhosis; etc.); (v) preventing, delaying, slowing,halting or ameliorating the cellular events of fibrosis (e.g.,preventing, delaying, slowing, halting or ameliorating increased matrixproduction, inhibition of matrix degradation, modulation of matrixreceptors to facilitate cell-matrix interactions, fibroblast activation,epithelial-to-mesenchymal transition, monocytic and lymphocytic cellinfiltration, and/or cell apoptosis); (vi) reducing interstitial diseasescore; (vii) preventing, delaying, ameliorating, slowing, halting orreducing myofibroblast accumulation and/or interstitial macrophageinfiltration; (viii) preventing, delaying, ameliorating, slowing,halting or reducing ECM gene transcription; (ix) preventing, slowing,halting or delaying the development of chronic kidney disease in at riskpatients; and/or (x) augmenting patient renal activity (e.g., glomerularfiltration rate).

The term “symptom(s)” as used herein, refers to common signs orindications that a patient is suffering from a specific condition ordisease. For example, chronic kidney disease-related symptomscontemplated herein include, but are not limited to, reduced glomerularfiltration rate; kidney damage; presence of protein, red and white bloodcells, bacteria, crystals and/or casts in urine; accumulation ofinterstitial macrophages, ECM accumulation; loss of nephrons; need tourinate frequently; increased water retention (puffiness or swelling) inthe legs, around the eyes, or in other parts of the body; high bloodpressure; anemia; loss of appetite, nausea and vomiting; itching; easybruising; pale skin; shortness of breath from fluid accumulation in thelungs; headaches; peripheral neuropathy; altered mental status(encephalopathy from the accumulation of waste products or uremicpoisons); chest pain due to pericarditis; bleeding (due to poor bloodclotting); bone pain and fractures; and abnormalities in kidney size.Pulmonary fibrosis-related symptoms contemplated herein include, but arenot limited to, dry unexplained cough, shortness of breath, anddiminished exercise tolerance. Liver fibrosis-related symptomscontemplated herein include, but are not limited to, yellowing of theskin (jaundice), fatigue, weakness, loss of appetite, itching, andbruising.

The term “effective amount,” as used herein, refers to the amountnecessary to elicit the desired biological response. In accordance withthe present embodiments, an effective amount of a cysteamine and/orcystamine product is the amount necessary to provide an observableeffect in at least one biological factor (e.g., improvement inglomerular filtration rate, improvement in cardiac output, improvementin blood oxygen level, etc.) for use in treating a biological condition(such as cirrhosis, pulmonary fibrosis, scleroderma, chronic kidneydisease, etc.). In one embodiment, an effective amount of a cysteamineand/or cystamine product is the amount necessary to prevent, slow, halt,or reduce progressive interstitial fibrosis in response to organ damage.In some embodiments, an effective amount delays, slows, halts or reducesthe rate of accumulation of ECM and/or ECM gene transcription in anorgan or tissue of a patient. The effective amount may include theamount necessary to delay, slow, or halt the progression of pulmonaryfibrosis, liver fibrosis, organ transplant fibrosis, and/or cardiacfibrosis. In some embodiments, an effective amount delays, slows, haltsor reduces the rate of loss of organ function. In some embodiments, aneffective amount delays, slows or halts the progression of CKD,improving glomerular filtration rate, reducing, delaying slowing,halting or preventing interstitial fibrosis in response to kidneyinjury, reducing, delaying slowing, halting or preventing ECMaccumulation or interstitial macrophage infiltration in the kidney,and/or reducing, delaying slowing, halting or preventing symptomsassociated with CKD. In some embodiments, the effective amount mayinclude the amount necessary to delay, slow, or halt the progression ofchronic kidney disease from Stage 1 to Stage 2, Stage 2 to Stage 3,Stage 3 to Stage 4, or Stage 4 to Stage 5. In certain embodiments, theeffective amount enables a 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, and 100% decreasein severity of complications associated with the biological condition(e.g., cardiovascular disease, anemia, hypertension and/or renalosteodystrophy, etc.). In some embodiments, an effective amount delays,slows, or halts the progression of pulmonary fibrosis, reducing,delaying slowing, halting or preventing ECM accumulation, reducing,slowing or halting the appearance of bullae, delaying, halting, orslowing reduction in diffusing capacity.

As used herein, reference to a “cysteamine product” includes cysteamine,the various cysteamine salts, which include pharmaceutically acceptablesalts of a cysteamine product, as well as prodrugs of cysteamine thatcan, for example, be readily metabolized in the body to producecysteamine. Also included within the scope of the present embodimentsare esters, amides, alkylated compounds, phosphorylated compounds,sulfated compounds, analogs, derivatives, conjugates, and metabolites ofcysteamine, which have the ability as described herein to ameliorateprogressive interstitial fibrosis and/or to delay, slow or halt theprogression of CKD. Also included within the scope of the presentembodiments are chemically modified forms cysteamine by such techniquesas labeling (e.g., with radionuclides or various enzymes), or covalentpolymer attachment such as pegylation (derivatization with polyethyleneglycol) or mixtures thereof. Various analogs, derivatives, conjugates,and metabolites of cysteamine are well known and readily used by thoseskilled in the art and include, for example, compounds, compositions andmethods of delivery as set forth in U.S. Pat. Nos. 6,521,266; 6,468,522;5,714,519; and 5,554,655 the disclosures of which are hereinincorporated by reference in their entirety. The disclosure is notlimited with respect to a specific cysteamine salt or ester orderivative. In some embodiments, cysteamine products include, but arenot limited to, hydrochloride salts, bitartrate salts, phosphorylatedderivatives, and sulfated derivatives. Examples of other cysteamineproducts include 2-aminopropane thiol-1,1-aminopropane thiol-2, N- andS-substituted cysteamine, AET, aminoalkyl derivatives, phosphorothioate,amifostine (U.S. Pat. No. 4,816,482). In one embodiment, a cysteamineproduct specifically excludes N-acetylcysteine. In one embodiment, acysteamine product specifically includes cystamine. In anotherembodiment, a cysteamine product specifically excludes cystamine.

As further contemplated herein, the advantages of cysteamine, as setforth herein, can be achieved by promoting the endogenous production ofcysteamine through natural metabolic process such as through the actionof co-enzyme A or as a metabolite of cysteine. This can be achieved by,for example, the administration of pantothenic acid. Pantothenic acid isa naturally occurring vitamin that is converted in mammals to coenzymeA, a substance vital to many physiological reactions. Cysteamine is acomponent of coenzyme A, thus increasing coenzyme A levels can result inincreased levels of circulating cysteamine. Alkali metal salts, such asmagnesium phosphate tribasic and magnesium sulphite (Epsom salts),enhance formation of coenzyme A. Furthermore, breakdown of coenzyme A tocysteamine is enhanced by the presence of a reducing agent, such ascitric acid. Thus, the combination of pantothenic acid and alkali metalsalts results in increased coenzyme A production and, concomitantly,cysteamine.

As used herein, reference to a “cystamine product” includes cystamine,the various cystamine salts, which include pharmaceutically acceptablesalts of a cystamine product, as well as prodrugs of cystamine that can,for example, be readily metabolized in the body to produce cystamine.Also included within the scope of the present embodiments are esters,amides, alkylated compounds, phosphorylated compounds, sulfatedcompounds, analogs, derivatives, conjugates, and metabolites ofcystamine, which have the ability as described herein to ameliorateprogressive interstitial fibrosis and/or to delay, slow or halt theprogression of CKD. Also included within the scope of the presentembodiments are chemically modified forms cystamine by such techniquesas labeling (e.g. with radionuclides or various enzymes), or covalentpolymer attachment such as pegylation (derivatization with polyethyleneglycol) or mixtures thereof. Various analogs, derivatives, conjugates,and metabolites of cystamine are well known and readily used by thoseskilled in the art. The disclosure is not limited with respect to aspecific cystamine salt or ester or derivative. In some embodiments,cystamine products include, but are not limited to, hydrochloride salts,bitartrate salts, phosphorylated derivatives, and sulfated derivatives.In one embodiment, a cystamine product specifically excludesN-acetylcysteine. In one embodiment, a cystamine product specificallyincludes cysteamine. In another embodiment, a cystamine productspecifically excludes cysteamine.

The term “pharmaceutically acceptable salt,” as used herein, refers toany salt of a cysteamine and/or cystamine product that ispharmaceutically acceptable and does not greatly reduce or inhibit theactivity of the cysteamine and/or cystamine product. Suitable examplesinclude acid addition salts, with an organic or inorganic acid such asacetate, tartrate, trifluoroacetate, lactate, maleate, fumarate,citrate, methane, sulfonate, sulfate, phosphate, nitrate, or chloride.

For human applications, an effective amount of a cysteamine and/orcystamine product of the present embodiments is used, optionally incombination with a pharmaceutically acceptable carrier. The compositionmay be dry, or it may be a solution. Treatment may be reactive, forcombating or preventing progression of an existing disease, orprophylactic, for preventing kidney damage in an organism susceptible todisease.

Several embodiments relate to a method of ameliorating progressiveinterstitial fibrosis in a mammal, comprising administering to themammal, for example a human, an effective amount of cysteamine and/orcystamine product. Several embodiments relate to a method of delaying,slowing, or halting the progression of CKD in a mammal, comprisingadministering to the mammal, for example a human, an effective amount ofcysteamine and/or cystamine product. Several embodiments relate to amethod of improving glomerular filtration rate in a mammal, comprisingadministering to the mammal, for example a human, an effective amount ofcysteamine and/or cystamine product. Several embodiments relate to amethod of improving liver function in a mammal, comprising administeringto the mammal, for example a human, an effective amount of cysteamineand/or cystamine product. Some embodiments relate to a method ofimproving cardiac output in a mammal, comprising administering to themammal, for example a human, an effective amount of cysteamine and/orcystamine product.

The cysteamine and/or cystamine product is administered in atherapeutically effective amount; typically, the composition is in unitdosage form. The amount of cysteamine and/or cystamine productadministered is dependent on the age, weight, and general condition ofthe subject, the severity of the condition being treated, and may bedetermined by the treating physician.

Suitable therapeutic amounts will be known to those skilled in the artand/or are described in the pertinent reference texts and literature.Current doses of cysteamine used to treat cystinosis are about 1.35 g/m2body surface area and are generally administered 4-times per day(Levtchenko et al., Pediatr Nephrol. 21:110-113, 2006). In someembodiments, a daily dose of about 0.01 mg to 1000 mg/kg body weight(BW) of a cysteamine and/or cystamine, or an equivalent molar quantityof a cysteamine and/or cystamine, is administered to an adult patient toelicit a desired response. In one aspect, the dose is administeredeither one time per day or multiple times per day. In some embodiments,cysteamine and/or cystamine may be administered one, two or three orfour or five times per day. In some embodiments, a daily dose of about10 mg to about 50 mg/kg BW of a cysteamine and/or cystamine product, oran equivalent molar quantity of a cysteamine and/or cystamine product,is administered to an adult patient to elicit a desired response. Insome embodiments, an effective dose may be about 0.5 mg/kg, 1 mg/kg, 5mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg/25 mg/kg, 30 mg/kg, 35 mg/kg, 40mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 70 mg/kg, 75 mg/kg, 80mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg,and may increase by 25 mg/kg increments up to 1000 mg/kg BW, or mayrange between any two of the foregoing values. In some embodiments,about 100 mg to 2 g of cysteamine and/or cystamine, or an equivalentmolar quantity thereof, is administered daily to an adult patient toelicit a desired response. In some embodiments, cysteamine and/orcystamine is administered at a total daily dose of from approximately0.25 g/m² to 4.0 g/m² body surface area. In some embodiments, a dose isadministered twice per day at about 0.5-1.0 g/m² (e.g., 0.7-0.8 g/m²)body surface area. In some embodiments, at least about 0.5, 0.6, 0.7,0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 g/m², orup to about 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2.0, 2.2, 2.5, 2.7, 3.0, or 3.5 g/m² may be administered at a totaldaily dose. In some embodiments, cysteamine and/or cystamine may beadministered at a total daily dose of about 1-1.5 g/m² body surfacearea, or 0.5-1 g/m2 body surface area, or about 0.7-0.8 g/m² bodysurface area, or about 1.35 g/m² body surface area. In some embodiments,doses of about 1.35 g/m² body surface area and are administered 4-5times per day. Salts or esters of the same active ingredient may vary inmolecular weight depending on the type and weight of the salt or estermoiety. In some embodiments, the daily dose is administered in multipledivided doses. In some embodiments, administration may continue for atleast 1 day, 5 days, 7 days, 14 days, 21 days, 1 month, 3 months, 6months, 9 months, 1 year, 2 years, or more.

In some embodiments, ½ to ⅛ of a maintenance dose of cysteamine and/orcystamine is administered to a patient initially and then the dose isgradually increased until the maintenance dose is reached. In someembodiments, a maintenance dose of 1.30 grams/m²/day of cysteamineand/or cystamine is administered in two, three, four, or five divideddoses. In some embodiments, a maintenance dose of 2.0 grams/day isadministered in two, three, four, or five divided doses.

For administration of the dosage form, e.g., a tablet or capsule orother oral dosage form comprising the enterically coated cysteamineand/or cystamine product, a total weight in the range of approximately100 mg to 1000 mg is used. In exemplary embodiments, the dosage form isorally administered to a patient suffering from kidney disease.Administration may continue for at least 3 weeks, 4 weeks, 6 weeks, 8weeks, 3 months, 6 months, 9 months, 1 year, 2 years, or more, or anytimeframe within the recited time limits.

Combination Therapy

In some embodiments, cysteamine and/or cystamine can be administered incombination with other therapies useful for treating fibrosis.Concurrent administration of two therapeutic agents does not requirethat the agents be administered at the same time or by the same route,as long as there is an overlap in the time period during which theagents are exerting their therapeutic effect. Simultaneous or sequentialadministration is contemplated, as is administration on different daysor weeks.

In some embodiments, cysteamine and/or cystamine can be administered incombination (either simultaneously in a single composition or inseparate compositions) with corticosteroids (such as prednisone) and/orother medications that suppress the body's immune system, such ascyclophosphamide, azathioprine, methotrexate, penicillamine, andcyclosporine.

In some embodiments, cysteamine and/or cystamine product can beadministered in combination (either simultaneously in a singlecomposition or in separate compositions) with inhibitors of therenin-angiotensin system. In some embodiments, cysteamine and/orcystamine product can be administered in combination (eithersimultaneously in a single composition or in separate compositions) withdrugs selected from the group consisting of renin inhibitors,angiotensin H antagonists, and angiotensin converting enzyme (ACE)inhibitors. Examples of renin inhibitors include, but are not limitedto, aliskiren and remikiren. Examples of angiotensin II antagonistsinclude, but are not limited to, losartan, irbesartan, olmesartan,candesartan, eprosartan, valsartan, and telmisartan. Examples of ACEinhibitors include, but are not limited to, AB-103, ancovenin,BRL-36378, BW-A575C, CGS-13928C, CL-242817, CV-5975, Equaten, EU-4865,EU-4867, EU-5476, foroxymithine, FPL 66564, FR-900456, Hoe-065, 15B2,indolapril, ketomethylureas, KRI-1177, KRI-1230, L-681176, libenzapril,MCD, MDL-27088, MDL-27467A, moveltipril, MS-41, nicotianamine,pentopril, phenacein, pivopril, rentiapril, RG-5975, RG-6134, RG-6027,RGH-0399, ROO-911, RS-10085-197, RS-2039, RS 5139, RS 86127, RU-44403,S-8308, SA-291, spiraprilat, SQ-26900, SQ-28084, SQ-28370, SQ-28940,SQ-31440, Synecor, utibapril, WF-10129, Wy-44221, Wy-44655, Y-23785,Yissum P-0154, zabicipril, Asahi Brewery AB-47, alatriopril, BMS 182657,Asahi Chemical C-111, Asahi Chemcal C-112, Dainippon DU-1777, mixanpril,Prentyl, zofenoprilat,1-(-(1-carboxy-6-(4-piperidinyl)hexyl)amino)-1-oxopropyloctahydro-1H-indole-2-carboxylic acid, Bioproject BP1.137, Chiesi CHF1514, Fisons FPL-66564, idrapril, Marion Merrell Dow MDL-100240,perindoprilat, Servier S-5590, alacepril, benazepril, captopril,cilazapril, delapril, enalapril, enalaprilat, fosinopril, fosinoprilat,imidapril, lisinopril, ramipril, perindopril, quinapril, saralasinacetate, temocapril, trandolapril, ceranapril, moexipril, quinaprilatand spirapril.

In some embodiments, cysteamine and/or cystamine product can beadministered in combination (either simultaneously in a singlecomposition or in separate compositions) with antioxidants such asglycyrrhizin, schisandra extract, ascorbic acid, glutathione, silymarin,lipoic acid, d-alpha-tocopherol, glycyrrhizin, ascorbic acid,glutathione, and vitamin B-complex. Alternatively, the combination oftherapeutics can be administered sequentially.

In some embodiments, cysteamine and/or cystamine product can beadministered in combination (either simultaneously in a singlecomposition or in separate compositions) with TGF-β antagonists. Theterm “TGF-β antagonist” and its cognates such as “inhibitor,”“neutralizing,” and “downregulating” refer to a compound (or itsproperty as appropriate), or other molecule, which acts as an antagonistof the biological activity of TGF-β. A TGF-β antagonist may, forexample, bind to and neutralize the activity of TGF-β; decrease TGF-βexpression levels; affect the stability or conversion of the precursormolecule to the active, mature form; interfere with the binding of TGF-βto one or more receptors; or it may interfere with intracellularsignaling of a TGF-β receptor. The term “direct TGF-β antagonist”generally refers to any compound that directly downregulates thebiological activity of TGF-β. A molecule “directly downregulates” thebiological activity of TGF-β if it downregulates the activity byinteracting with a TGF-β gene, a TGF-β transcript, a TGF-β ligand, or aTGF-β receptor. Examples of TGF-β antagonists that may be used includebut are not limited to monoclonal and polyclonal antibodies directedagainst one or more isoforms of TGF-β (U.S. Pat. No. 5,571,714; WO97/13844; and WO 00/66631; WO 05/097832; WO 05/101149; WO 06/086469);dominant negative and soluble TGF-(3 receptors or antibodies directedagainst TGF-β receptors (Flavell et al., Nat. Rev. Immunol. 2:46-53(2002); U.S. Pat. No. 5,693,607; U.S. Pat. No. 6,001,969; U.S. Pat. No.6,008,011; U.S. Pat. No. 6,010,872; WO 92/00330; WO 93/09228; WO95/10610; and WO 98/48024; LAP (WO 91/08291); LAP-associated TGF-β (WO94/09812); TGF-β-binding glycoproteins/proteoglycans such as fetuin(U.S. Pat. No. 5,821,227); decorin, betaglycan, fibromodulin, lumican,and endoglin (U.S. Pat. No. 5,583,103; U.S. Pat. No. 5,654,270; U.S.Pat. No. 5,705,609; U.S. Pat. No. 5,726,149; U.S. Pat. No. 5,824,655;U.S. Pat. No. 5,830,847; U.S. Pat. No. 6,015,693; WO 91/04748; WO91/10727; WO 93/09800; and WO 94/10187); mannose-6-phosphate ormannose-1-phosphate (U.S. Pat. No. 5,520,926); prolactin (WO 97/40848);insulin-like growth factor 11 (WO 98/17304); extracts of plants, fungi,and bacteria (EU 813875; JP 8119984; and U.S. Pat. No. 5,693,610);antisense oligonucleotides (U.S. Pat. No. 5,683,988; U.S. Pat. No.5,772,995; U.S. Pat. No. 5,821,234; U.S. Pat. No. 5,869,462; and WO94/25588); and any mutants, fragments, or derivatives of theabove-identified molecules that retain the ability to inhibit thebiological activity of TGF-β. Numerous small molecule TGF-β antagoniststhat may be useful are also well known to those of skill in the art,including, but not limited to, those described in WO 02/62753; WO02/62776; WO 02/62787; WO 02/62793; WO 02/62794; WO 02/66462; WO02/94833; WO 03/87304; WO 03/97615; WO 03/97639; WO 04/10929; WO04/21989; WO 04/22054; WO 04/24159; WO 04/26302; WO 04/26871; U.S. Pat.No. 6,184,226; WO 04/16606; WO 04/47818; WO 04/48381; WO 04/48382; WO04/48930; WO 04/50659; WO 04/56352; WO 04/72033; WO 04/87056 WO05/10049; WO 05/032481; WO 05/065691; WO 05/92894; WO 06/026305; WO06/026306; and WO 06/052568.

In some embodiments, cysteamine and/or cystamine product can beadministered in combination (either simultaneously in a singlecomposition or in separate compositions) with tumor necrosis factor(TNF)-α antagonists. The term “TNF-α antagonist” and its cognates suchas “inhibitor,” “neutralizing,” and “downregulating” refer to a compound(or its property as appropriate), or other molecule, which acts as anantagonist of the biological activity of TNF-α. A TNF-α antagonist may,for example, bind to and neutralize the activity of TNF-α; decreaseTNF-α expression levels; interfere with the binding of TNF-α to one ormore receptors; or it may interfere with intracellular signaling of aTNF-α receptor. The term “direct TNF-α antagonist” generally refers toany compound that directly downregulates the biological activity ofTNF-α. A molecule “directly downregulates” the biological activity ofTNF-α if it downregulates the activity by interacting with a TNF-α gene,a TNF-α transcript, a TNF-α ligand, or a TNF-α receptor. Examples ofTNF-α antagonists include, but not limited to, a TNF chemical or proteinantagonist, TNF monoclonal or polyclonal antibody or fragment, a solubleTNF receptor (e.g., p55, p70 or p85) or fragment, fusion polypeptidesthereof, or a small molecule TNF antagonist, e.g., TNF binding protein Ior II (TBP-1 or TBP-11), nerelimonmab, infliximab, etanercept, CDP-571,CDP-870, afelimomab, lenercept, and the like.

It is further contemplated that the cysteamine and/or cystaminecomposition is administered with a second agent useful for treatingkidney fibrosis. A second agent may be other therapeutic agents, such asanti-diabetic agents, cytokines, growth factors, other anti-inflammatoryagents, anti-coagulant agents, agents that will lower or reduce bloodpressure, agents that will reduce cholesterol, triglycerides, LDL, VLDL,or lipoprotein(a) or increase HDL, agents that will increase or decreaselevels of cholesterol-regulating proteins, anti-neoplastic drugs ormolecules.

Exemplary second agents include, but are not limited to, agents used totreat diabetes, cyclophosphamide, either alone or in combination withmycophenolate mofetil (MMF) or prednisolone, or other corticosteroids,anti-inflammatory agents, azathioprine, IFN-gamma.

Exemplary anti-diabetic agents include, but are not limited to, 1)sulfonylureas (e.g., glimepiride, glisentide, sulfonylurea, AY31637); 2)biguanides (e.g., 3) alpha-glucosidase inhibitors (e.g., acarbose,miglitol); 4) thiazol-idinediones (e.g., troglitazone, pioglitazone,rosiglitazone, glipizide, balaglitazone, rivoglitazone, netoglitazone,troglitazone, englitazone, AD 5075, T 174, YM 268, R 102380, NC 2100,NIP 223, NIP 221, MK 0767, ciglitazone, adaglitazone, CLX 0921,darglitazone, CP 92768, BM 152054); 5) glucagon-like-peptides (GLP) andGLP analogs or agonists of GLP-1 receptor (e.g., exendin) or stabilizersthereof (e.g., DPP4 inhibitors, such as sitagliptin); and 6) insulin oranalogues or mimetics thereof (e.g., LANTUS®).

Additional anti-fibrotic agents contemplated for use in the methods ofthe present disclosure can be any agent that affects fibrosis.Contemplated agents include, but are not limited to, those that reducethe activity of transforming growth factor-beta (TGF-β) (including butnot limited to GC-1008 (Genzyme/MedImmune); lerdelimumab (CAT-152;Trabio, Cambridge Antibody); metelimumab (CAT-192, Cambridge Antibody,);LY-2157299 (Eli Lilly); ACU-HTR-028 (Opko Health)) including antibodiesthat target one or more TGF-isoforms, inhibitors of TGF-β receptorkinases TGFBR1 (ALK5) and TGFBR2, and modulators of post-receptorsignaling pathways; chemokine receptor signaling; endothelin receptorantagonists including inhibitors that target both endothelin receptor Aand B and those that selectively target endothelin receptor A (includingbut not limited to ambrisentan; avosentan; bosentan; clazosentan;darusentan; BQ-153; FR-139317, L-744453; macitentan; PD-145065;PD-156252; PD163610; PS-433540; S-0139; sitaxentan sodium; TBC-3711;zibotentan); agents that reduce the activity of connective tissue growthfactor (CTGF) (including but not limited to FG-3019, FibroGen), and alsoincluding other CTGF-neutralizing antibodies; matrix metalloproteinase(MMP) inhibitors (including but not limited to MMPI-12, PUP-1 andtigapotide triflutate); agents that reduce the activity of epidermalgrowth factor receptor (EGFR) including but not limed to erlotinib,gefitinib, BMS-690514, cetuximab, antibodies targeting EGF receptor,inhibitors of EGF receptor kinase, and modulators of post-receptorsignaling pathways; agents that reduce the activity of platelet derivedgrowth factor (PDGF) (including but not limited to Imatinib mesylate(Novartis)) and also including PDGF neutralizing antibodies, antibodiestargeting PDGF receptor (PDGFR), inhibitors of PDGFR kinase activity,and post-receptor signaling pathways; agents that reduce the activity ofvascular endothelial growth factor (VEGF) (including but not limited toaxitinib, bevacizumab, BIBF-1120, CDP-791, CT-322, IMC-18F1, PTC-299,and ramucirumab) and also including VEGF-neutralizing antibodies,antibodies targeting the VEGF receptor 1 (VEGFR1, Flt-1) and VEGFreceptor 2 (VEGFR2, KDR), the soluble form of VEGFR1 (sFlt) andderivatives thereof which neutralize VEGF, and inhibitors of VEGFreceptor kinase activity; inhibitors of multiple receptor kinases suchas BIBF-1120 which inhibits receptor kinases for vascular endothelialgrowth factor, fibroblast growth factor, and platelet derived growthfactor; agents that interfere with integrin function (including but notlimited to STX-100 and IMGN-388) and also including integrin targetedantibodies; agents that interfere with the pro-fibrotic activities ofIL-4 (including but not limited to AER-001, AMG-317, APG-201, andsIL-4Rα) and IL-13 (including but not limited to AER-001, AMG-317,anrukinzumab, CAT-354, cintredekin besudotox, MK-6105, QAX-576, SB-313,SL-102, and TNX-650) and also including neutralizing anti-bodies toeither cytokine, antibodies that target IL-4 receptor or IL-13 receptor,the soluble form of IL-4 receptor or derivatives thereof that isreported to bind and neutralize both IL-4 and IL-13, chimeric proteinsincluding all or part of IL-13 and a toxin particularly pseudomonasendotoxin, signaling though the JAK-STAT kinase pathway; agents thatinterfere with epithelial mesenchymal transition including inhibitors ofmTor (including but not limited to AP-23573); agents that reduce levelsof copper such as tetrathiomolybdate; agents that reduce oxidativestress including N-acetyl cysteine and tetrathiomolybdate; andinterferon gamma. Also contemplated are agents that are inhibitors ofphosphodiesterase 4 (PDE4) (including but not limited to Roflumilast);inhibitors of phosphodiesterase 5 (PDE5) (including but not limited tomirodenafil, PF-4480682, sildenafil citrate, SLx-2101, tadalafil,udenafil, UK-369003, vardenafil, and zaprinast); or modifiers of thearachidonic acid pathway including cyclooxygenase and 5-lipoxegenaseinhibitors (including but not limited to Zileuton). Further contemplatedare compounds that reduce tissue remodeling or fibrosis including prolylhydrolase inhibitors (including but not limited to 1016548, CG-0089,FG-2216, FG-4497, FG-5615, FG-6513, fibrostatin A (Takeda), lufironil,P-1894B, and safironil) and peroxisome proliferator-activated receptor(PPAR)-gamma agonists. (including but not limited to pioglitazone androsiglitazone).

Other specific anti-fibrotic agents contemplated include relaxin,pirfenidone, ufironil, surifonil, a TGF-β antibody, CAT-192, CAT-158;ambresentan, thelin; FG-3019, a CTGF antibody; anti-EGFR antibody; aEGFR kinase inhibitor; tarceva; gefitinib; PDGF antibody, PDGFR kinaseinhibitor; gleevec; BIBF-1120, VEGF, FGF, and PDGF receptor inhibitor;anti-integrin antibody; IL-4 antibody; tetrathiomolybdate, a copperchelating agent; interferon-gamma; NAC, a cysteine pro-drug; hepatocytegrowth factor (HGF); KGF; angiotension receptor blockers, ACEinhibitors, rennin inhibitors; COX and LO inhibitors; Zileuton;monteleukast; avastin; statins; PDE5 inhibitors, such as sildenafil,udenafil, tadalafil, vardenafil, or zaprinast; rofumilast; etanercept(Enbrel); procoagulant; prostaglandins, such as PGE2, PRX-08066, a 5HT2Breceptor antagonist; cintredekin besudotox, a chimeric human IL13conjugated to a genetically engineered Pseudomonas exotoxin;roflumilast, a PDE4 inhibitor; FG-3019, an anti-connective tissue growthfactor human monoclonal antibody; GC-1008, a TGF-β human monoclonalantibody; treprostinil, a prostacyclin analog; interferon-α; QAX-576, aIL13 modulator; WEB 2086, a PAF-receptor antagonist; imatinib mesylate;FG-1019; Suramin; Bosentan; IFN-1b; anti-IL-4; anti-IL-13; taurine,niacin, NF-κB antisense oligonucleotides; and nitric oxide synthaseinhibitors.

It is contemplated the cysteamine composition and the second agent maybe given simultaneously, in the same formulation. It is furthercontemplated that the agents are administered in a separate formulationand administered concurrently, with concurrently referring to agentsgiven within 30 minutes of each other.

In another aspect, the second agent is administered prior toadministration of the cysteamine and/or cystamine. Prior administrationrefers to administration of the second agent within the range of oneweek prior to treatment with cysteamine, up to 30 minutes beforeadministration of cysteamine. It is further contemplated that the secondagent is administered subsequent to administration of the cysteamineand/or cystamine composition. Subsequent administration is meant todescribe administration from 30 minutes after cysteamine and/orcystamine treatment up to one week after cysteamine and/or cystamineadministration.

It is further contemplated that other adjunct therapies may beadministered, where appropriate. For example, the patient may also beadministered a diabetic diet or food plan, surgical therapy, orradiation therapy where appropriate.

Formulation and Delivery

The cysteamine and/or cystamine product of the present embodiments maybe formulated into compositions together with pharmaceuticallyacceptable carriers for parenteral injection, for oral administration insolid or liquid form, for rectal administration, and the like. Thecysteamine and/or cystamine product may be administered orally(including buccal, sublingual, inhalation), nasally, rectally,vaginally, intravenously, intradermally, subcutaneously and topically.Cysteamine and/or cystamine product may be formulated into compositionssuitable for administration for example with suitable carriers,diluents, thickeners, adjuvants, etc., as are routine in the formulationart. Compositions of the present embodiments may also include additionalactive ingredients. Dosage forms include solutions, powders, tablets,capsules, gel capsules, suppositories, topical ointments and creams andaerosols for inhalation.

In one embodiment, administration is performed at the site of affectedtissue needing treatment by direct injection into the site or via asustained delivery or sustained release mechanism, which can deliver theformulation internally. For example, biodegradable microspheres orcapsules or other biodegradable polymer configurations capable ofsustained delivery of a composition (e.g., a soluble polypeptide,antibody, or small molecule) can be included in the formulations of theinvention implanted at the site.

In some embodiments, cysteamine and/or cystamine product is administeredvia oral delivery. Compositions for oral administration include powdersor granules, suspensions or solutions in water or non-aqueous media,oil-in-water emulsions or water-in-oil liquid emulsions, capsules,sachets, troches, tablets or SECs (soft elastic capsules or caplets).Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids,carrier substances of binders may be desirably added to suchformulations. The use of such formulations has the effect of deliveringthe cysteamine and/or cystamine to the alimentary canal for exposure tothe mucosa thereof. Accordingly, the formulation can consist of materialeffective in protecting the product from pH extremes of the stomach, orin releasing the product over time, to optimize the delivery thereof toa particular mucosal site. A tablet may be made by compression ormolding, optionally with one or more accessory ingredients. The tabletsmay optionally be coated or scored and may be formulated so as toprovide slow or controlled release of the active ingredients therein.

Compositions may be formulated in a conventional manner using additionalpharmaceutically acceptable carriers or excipients as appropriate. Thus,the composition may be prepared by conventional means with additionalcarriers or excipients such as binding agents (e.g., pregelatinisedmaize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose);filters (e.g., lactose, microcrystalline cellulose or calcium hydrogenphosphate); lubricants (e.g., magnesium stearate, talc or silica);disintegrates (e.g., starch or sodium starch glycolate); or wettingagents (e.g., sodium lauryl sulfate). Tablets may be coated by methodswell known in the art. The preparations may be also contain flavoring,coloring and/or sweetening agents as appropriate.

Pharmaceutical formulations, which may conveniently be presented in unitdosage form, may be prepared according to conventional techniques wellknown in the pharmaceutical industry. Such techniques include the stepof bringing into association the active ingredients with thepharmaceutical carrier(s) or excipient(s). In general the formulationsare prepared by uniformly and intimately bringing into association theactive ingredients with liquid carriers or finely divided soled carriersor both, and then, if necessary, shaping the product.

Delayed or Controlled Release Dosage Forms

In some embodiments, the cysteamine and/or cystamine product is adelayed or controlled release dosage form. The preparation of delayed,controlled or sustained/extended release forms of pharmaceuticalcompositions with the desired pharmacokinetic characteristics is knownin the art and can be accomplished by a variety of methods. For example,oral controlled delivery systems include dissolution-controlled release(e.g., encapsulation dissolution control or matrix dissolution control),diffusion-controlled release (reservoir devices or matrix devices), ionexchange resins, osmotic controlled release or gastroretentive systems.Dissolution controlled release can be obtained, e.g., by slowing thedissolution rate of a drug in the gastrointestinal tract, incorporatingthe drug in an in soluble polymer, and coating drug particles orgranules with polymeric materials of varying thickness. Diffusioncontrolled release can be obtained, e.g., by controlling diffusionthrough a polymeric membrane or a polymeric matrix. Osmoticallycontrolled release can be obtained, e.g., by controlling solvent influxacross a semipermeable membrane, which in turn carries the drug outsidethrough a laser-drilled orifice. The osmotic and hydrostatic pressuredifferences on either side of the membrane govern fluid transport.Prolonged gastric retention may be achieved by, e.g., altering densityof the formulations, bioadhesion to the stomach lining, or increasingfloating time in the stomach. For further detail, see the Handbook ofPharmaceutical Controlled Release Technology, Wise, ed., Marcel Dekker,Inc., New York, N.Y. (2000), incorporated by reference herein in itsentirety, e.g., Chapter 22 (“An Overview of Controlled ReleaseSystems”).

The concentration of cysteamine and/or cystamine product in theseformulations can vary widely, for example from less than about 0.5%,usually at or at least about 1% to as much as 15 or 20% by weight andare selected primarily based on fluid volumes, manufacturingcharacteristics, viscosities, etc., in accordance with the particularmode of administration selected. Actual methods for preparingadministrable compositions are known or apparent to those skilled in theart and are described in more detail in, for example, Remington'sPharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa.(1980).

In certain embodiments, the delayed or controlled release form isenterically coated. An enterically coated drug or tablet refers,generally, to a drug or tablet that is coated with a substance (an“enteric coating”) that remains intact or substantially intact such thatthe drug or tablet is passed through the stomach but dissolves andreleases the drug in the small intestine. Enterically coated cysteamineproducts are described in International Patent Publication Nos. WO07/089,670 and WO 09/070,781, hereby incorporated by reference in theirentirety.

Briefly, an enteric coating can be a polymer material or materials whichencase a medicament core (e.g., cystamine, cysteamine, CYSTAGON™ orother cysteamine and/or cystamine product). Typically a substantialamount or all of the enteric coating material is dissolved before themedicament or therapeutically active agent is released from the dosageform, so as to achieve delayed dissolution or delivery of the medicamentcore. A suitable pH-sensitive polymer is one which will dissolve inintestinal environment at a higher pH level (pH greater than 4.5), suchas within the small intestine, and therefore permit release of thepharmacologically active substance in the regions of the small intestineand not in the upper portion of the GI tract, such as the stomach.Enteric coatings for acid-resistant tablets, capsules and capletsinclude, but are not limited to, acetate phthalate, propylene glycol andsorbitan monoleate.

For administration of the dosage form, e.g., a tablet or capsule orother oral dosage form comprising the enterically coated cysteamineand/or cystamine product, a total weight in the range of approximately100 mg to 1000 mg is used. In exemplary embodiments, the dosage form isorally administered to a patient suffering from kidney disease.Administration may continue for at least 3 weeks, 4 weeks, 6 weeks, 8weeks, 3 months, 6 months, 9 months, 1 year, 2 years, or more, or anytimeframe within the recited time limits.

The following Examples are presented for the purposes of illustrationand should not be construed as limitations.

Example 1 IP Cysteamine HCl Dose Trial

Unilateral ureteral obstruction (UUO) was performed on ten mice.Starting on the day of UUO, two of the mice were given dailyintraperitoneal (IP) injections of phosphate buffered saline (PBS), twoof the mice were given daily intraperitoneal (IP) injections of 100mg/kg freshly mixed cysteamine HCL in PBS, and two of the mice weregiven daily intraperitoneal (IP) injections of 200 mg/kg freshly mixedcysteamine HCL in PBS. One group of mice (n=2) was given dailyintraperitoneal (IP) injections of freshly mixed cysteamine HCL in PBSstarting at a dose of 200 mg/kg on the day of UUO and the dose wasgradually increased every two days to a maximum dose of 400 mg/kg.Another group of mice (n=2) was given daily intraperitoneal (IP)injections of 200 mg/kg freshly mixed cysteamine HCL in PBS starting 5days after UUO (D5 200 mg/kg). Possible seizures and somnolence wereobserved in mice given cysteamine HCL immediately after surgery andseveral mice died at high dose levels. Surviving mice were sacrificed atday 14 after UUO and total renal collagen levels (μg hydroxyproline/mg)were measured. The results of the IP cysteamine HCL dose trials areshown in FIG. 4. At day 14, the 400 mg/kg dose group exhibited decreasedtotal collagen levels compared to placebo (PBS).

Example 2 Oral Cystagon® Dose Trial

UUO was performed on sixteen mice and placebo (n=4 mice) or 200 mg/kgcysteamine bitartrate dissolved in drinking water administered on thefirst day after UUO. It was assumed that an average 25 g mouse drinks 5mLs of water per day; cysteamine bitartrate was diluted in water andprepared daily to provide 200 mg/kg, 400 mg/kg or 600 mg/kg. One groupof mice (n=4) received 200 mg/kg cysteamine bitartrate dissolved indrinking water for fourteen days. In one group of mice (n=4), the doseof cysteamine bitartrate dissolved in the drinking water was increasedevery 2 days to a maximum of 400 mg/kg. In one group of mice (n=4), thedose of cysteamine bitartrate dissolved in the drinking water wasincreased every 2 days to a maximum of 600 mg/kg. No deaths or abnormalbehavior was observed, even at high doses. Mice were sacrificed at day14 after UUO and total renal collagen levels (μg hydroxyproline/mg) weremeasured. Total collagen in the tissue was calculated on the assumptionthat collagen contains 12.7% hydroxyproline by weight. As shown in FIG.5, the severity of fibrosis as measured by total collagen content wasattenuated in mice receiving 400 mg/kg and 600 mg/kg cysteaminebitartrate dissolved in drinking water.

Example 3 Cysteamine Treatment Reduces Severity of Fibrosis in UUOKidneys

Unilateral ureteral obstruction (UUO) was performed on 8 week-oldwild-type male mice on a C57BL6 background. The degree of renal fibrosiswas investigated using two doses of cysteamine bitartrate: 400 and 600mg/kg/day. Cysteamine bitartrate was added to drinking water that wasfreshly made every 24 h starting on the first day after UUO at 200 mg/kgand the dose was increased every 2 days until the maximum dosage wasreached. It was assumed that an average 25 g mouse drinks 5 mLs of waterper day; cysteamine bitartrate was diluted in water and prepared dailyto provide 400 mg/kg or 600 mg/kg. The control group did not receivecysteamine bitartrate treatment. Groups of mice (n=8-10 mice per groupat each time-point) were studied 3, 7, 14, and 21 days after the onsetof chronic injury induced by UUO.

Total collagen was measured as hydroxyproline concentration inhydrolysates extracted from frozen normal (unobstructed) and UUO kidneysamples. Total collagen in the tissue was calculated on the assumptionthat collagen contains 12.7% hydroxyproline by weight. Both doses, 400and 600 mg/kg/day, showed statistically significant reductions (P<0.01)in kidney collagen levels by day 14. Compared to the untreated group,mice receiving 600 mg/kg/day cysteamine bitartrate showed a 21 percentdecrease in collagen levels by day 14 and a 25 percent decrease incollagen levels at day 21 after UUO (FIG. 6). Collagen reduction wasconfirmed by sirius red staining.

Example 4 Cysteamine Decreases ECM Gene Transcription in UUO Kidneys ina Dose Dependent Manner

One mechanism by which cysteamine treatment is believed to slow or haltfibrosis is through reduction of ECM synthesis rates.

Unilateral ureteral obstruction (UUO) was performed on 8 week-oldwild-type male mice on a C57BL6 background. Cysteamine bitartrate wasadded to the drinking water daily starting on the first day after UUO at200 mg/kg and the dose was increased every 2 days to a maximum dose ofeither 400 mg/kg (n=6-8 mice) or 600 mg/kg (n=6-8 mice). It was assumedthat an average 25 g mouse drinks 5 mLs of water per day; cysteaminebitartrate was diluted in water and prepared daily to provide 400 mg/kgor 600 mg/kg. The control group did not receive cysteamine bitartratetreatment. The kidneys were harvested at 3, 7, and 14 days after UUO.

Extracellular matrix (ECM) gene mRNA levels were measured bysemiquantitative real-time PCR (qPCR) in both doses at 3, 7 and 14 daysafter UUO. Real-time qPCR) was performed with specific primers forcollagen 1A1, collagen 3A1, fibronectin, and other profibrotic andproinflammatory genes using standard protocols. Semi-quantitativereal-time qPCR data was analyzed with REST software using both GAPDH and18S as reference genes. Reactions were run in triplicate, and meanthreshold crossing cycle (Ct) values were compared. ‡ P<0.05; † P<0.01.

ECM gene transcription levels were significantly down-regulated in UUOkidneys of cysteamine-treated mice. Procollagen I mRNA levels were 56percent lower in the mice treated with 600 mg/kg at day 14. At day 7after UUO, despite no difference in total collagen (See Example 3 andFIG. 6), there was a nearly 40 percent reduction in kidney fibronectinand procollagen III mRNA levels in mice treated with 400 mg/kg/daycysteamine bitartrate and a nearly 60 percent reduction in fibronectin,procollagen I and procollagen III at higher doses of cysteaminebitartrate (600 mg/kg/day). Results are summarized in FIG. 7.

Example 5 Cysteamine Decreases Myofibroblast Activation and Accumulation

Myofibroblast activation and accumulation was examined after UUO bymeasuring the expression of α-smooth muscle actin (α-SMA), a marker ofmyofibroblast activation. Interstitial myofibroblasts are the primarymatrix-producing cell in response to kidney injury.

UUO was performed on 8 week-old wild-type male mice on a C57BL6background. Cysteamine bitartrate was added to the drinking water dailystarting on the first day after UUO at 200 mg/kg and the dose wasincreased every 2 days to a maximum dose of either 400 mg/kg (n=6-8mice) or 600 mg/kg (n=6-8 mice). It was assumed that an average 25 gmouse drinks 5 mLs of water per day; cysteamine bitartrate was dilutedin water and prepared daily to provide 400 mg/kg or 600 mg/kg. Thecontrol group did not receive cysteamine bitartrate treatment. Animalswere injected with 50 mg/kg of BrdU at 10 mg/mL the day prior tosacrifice. Kidneys were harvested at days 7 and 14 after UUO. Cellproliferation was measured by counting BrdU positive cells using amonoclonal anti-BrdU antibody. Myofibroblast recruitment was quantifiedafter immunoperoxidase staining using peroxidase-conjugated murineanti-human α-smooth muscle actin (α-SMA) 1A4 monoclonal antibody(Sigma):

Statistically significant reductions in the numbers of α-SMA-positivemyofibroblasts were observed for both doses of cysteamine at day 7 andday 14 after UUO (P<0.01). The largest reductions of 38% and 47% wereseen at day 7 in the 400 mg/kg and 600 mg/kg groups, respectively. Atday 14, the 400 mg/kg dosage group exhibited a 24% reduction inα-SMA-positive myofibroblasts and the 600 mg/kg dosage group exhibited a33% reduction in α-SMA-positive myofibroblasts. See FIG. 8.

Example 6 Cysteamine Blocks Interstitial Macrophage Infiltration atAdvanced Time Points

UUO was performed on 8 week-old wild-type male mice on a C57BL6background. Cysteamine bitartrate was added to the drinking water dailystarting at a dose of 200 mg/kg on the first day after UUO and dose wasincreased every 2 days to a maximum dose of either 400 mg/kg (n=4-8mice) or 600 mg/kg (n=5-8 mice). It was assumed that an average 25 gmouse drinks 5 mLs of water per day; cysteamine bitartrate was dilutedin water and prepared daily to provide 400 mg/kg or 600 mg/kg. Thecontrol group (n=5-8 mice) did not receive cysteamine bitartratetreatment. The kidneys (obstructed and contralateral) were harvested atday 7 and day 14 after UUO. Macrophage accumulation was analyzed byconfocal staining using F4/80 rat anti-mouse macrophage monoclonalantibody (Serotec Ltd., Oxford, UK).

Computed-assisted image analysis of immunohistochemically stained kidneysections at day 14 UUO show significantly less interstitial inflammationas measured by the number of F4/80+ interstitial macrophages andsignificantly fewer interstitial myofibroblasts. FIG. 9 showsrepresentative confocal images (400×) of F4/80-positive interstitialmacrophages and a graph summarizing the results of semi-quantitativeanalysis of F4/80-positive interstitial area. (n=4-8/group; † P<0.01). Asignificant reduction (34%) in interstitial macrophage infiltration wasobserved at day 14 in mice treated with 600 mg/kg/day. No difference ininterstitial macrophage infiltration was seen at day 7.

Example 7 Cysteamine Bitartrate Modulates Profibrotic Signaling atAdvanced Timepoints

The expression patterns of pro-inflammatory and pro-fibrotic cytokineswere investigated at day 7 and day 14 in total kidney homogenate bysemi-quantitative real time qPCR.

UUO was performed on 8 week-old wild-type male mice on a C57BL6background. Cysteamine bitartrate was added to the drinking water dailystarting at a dose of 200 mg/kg on the first day after UUO and the dosewas increased every 2 days to a maximum dose of either 400 mg/kg (n=6-8mice) or 600 mg/kg (n=6-8 mice). It was assumed that an average 25 gmouse drinks 5 mLs of water per day; cysteamine bitartrate was dilutedin water and prepared daily to provide 400 mg/kg or 600 mg/kg. Thecontrol group (n=5-8 mice) did not receive cysteamine bitartratetreatment. The kidneys (obstructed and contralateral) were harvested atday 7 and day 14 after UUO.

Semi-quantitative real-time PCR was performed with specific primers toTGF-β and TGF-β receptor 1 genes using iCycler (Bio-Rad) with standardprotocol. Relative mRNA transcription levels of TGF-β and TGF-β receptor1 genes in UUO kidneys of cysteamine treated mice were determined withrespect to control mice. Semi-quantitative real-time PCR data wasanalyzed with REST software using both GAPDH and 18S as reference genes.Each sample was performed in triplicate. (‡ P<0.05, † P<0.01).

At day 7 after UUO, expression of both the profibrotic cytokine TGF-βand the TGF-β receptor were significantly up-regulated by approximately60 percent in mice treated with high doses of cysteamine bitartratecompared to control mice (P<0.01). This suggests that thedown-regulation of ECM gene transcription observed at day 7 is TGF-βindependent. At day 14, however, the mRNA levels of TGF-β and the TGF-βreceptor were significantly down-regulated by 47 percent and 64 percentin mice treated with 400 mg/kg or 600 mg/kg cysteamine bitartrate,respectively, compared to control mice (P<0.05). No difference wasobserved in the expression levels of IL-1β, TNF-α, IL-6, Gal-3, andEndo180 at day 14. See Table 2 and FIG. 10.

TABLE 2 Expression of TGF-β and the TGF-β receptor After UUO inCysteamine Treated Mice Cystagon Cystagon Cystagon Cystagon 400 mg/kg600 mg/kg 400 mg/kg 600 mg/kg Day 7 Day 7 Day 14 Day 14 TGF-β 0.52 ±0.13 0.74 ± 0.19** 0.61 ± 0.21 0.66 ± 0.22  TGF-β 0.72 ± 0.30 1.38 ±0.36** 0.56 ± 0.22 0.42 ± 0.13** receptor

Example 8 Cysteamine Modulates Antioxidant Status at Early Timepoints

In order to investigate the importance of total redox status duringfibrotic injury, total kidney thiol content was measured as an indicatorof antioxidant status.

UUO was performed on 8 week-old wild-type male mice on a C57BL6background (n=5/group). Cysteamine bitartrate was added to the drinkingwater daily starting at a dose of 200 mg/kg on the first day after UUOand the dose was increased every 2 days to a maximum dose of either 400mg/kg or 600 mg/kg. It was assumed that an average 25 g mouse drinks 5mLs of water per day; cysteamine bitartrate was diluted in water andprepared daily to provide 400 mg/kg or 600 mg/kg. The control group didnot receive cysteamine bitartrate treatment. The kidneys (obstructed andcontralateral) were harvested at days 7, 14 and 21 after UUO. Tissuefrom contralateral and UUO kidneys was processed in antioxidant bufferand analyzed for total thiol content fluorometrically and normalized toprotein content using a Measure-iT™ Thiol Assay Kit (Invitrogen).

Total kidney thiol content in UUO tissue was significantly decreased 40percent compared to the contralateral kidney in control dose mice(contralateral vs. UUO, n=5-6/group: 1397 vs. 838 mM thiol, P<0.01). Atday 7 after UUO, total kidney thiol content remained, at levels close tothat of the contralateral kidney in the 400 mg/kg or 600 mg/kgcysteamine-treated mice compared to control (FIG. 11). In addition,there was no difference between cysteamine-treated and control mice inthe modulation of the expression of the oxidant and anti-oxidant genesNox2, Nox4, and SOD1 as determined by semi-quantitative real time qPCRat day 7 and day 14 (data not shown).

Example 9 Evaluation of the Role of Endogenous Cysteamine Sythesized byan Enzamatic Pathway Encoded by the Vanin-1 Gene

The role of endogenous cysteamine synthesized via an enzymatic pathwaythat is encoded by the vanin gene in protection against fibrosis wasexamined by comparing the degree of fibrosis between littermate controls(Vanin+/+) and Vanin−/− mice. UUO was performed on 8 week-old Vanin+/+(n=3-7) and Vanin−/− (n=3-6) mice. The kidneys (obstructed andcontralateral (NK)) were harvested at days 14 and 21 after UUO and totalrenal collagen levels (μg hydroxyproline/mg) were measured. See FIG.12C.

No statistically significant difference in total collagen levels wasobserved in the UUO kidneys of Vanin+/+ and Vanin−/− at 14 days afterUUO. There was, however, a non-significant trend toward more fibrosis inthe Vnn1−/− UUO kidneys at advanced stages (day 21 after UUO). TheVanin−/− strain used for these studies was not in a pure C57BL/6background and the values for total collagen were much lower thantypically seen in C57BL/6 mice. Backcrossing of the Vanin−/− mutationinto a pure C57BL/6 background will be performed to determine if theobserved differences can be accentuated.

In order to test whether the rate of progression of the renal phenotypein Cystinosin deficient (Ctns−/−) mice is attenuated by the endogenouscysteamine synthesized via a vanin-1 mediated enzymatic pathway, acolony of Ctns−/− Vanin-1−/− double knock-out mice was generated. Ratesof polyuria significantly increased in Ctns−/− Vanin-1−/− knock-out miceat 6 months of age; however, the difference diminished at 9 months andthere was no difference in kidney weight of the double knock-out micecompared to heterozygous controls. There was a significant increase inblood urea nitrogen (BUN) in the double knock-out mice compared toheterozygous controls at 10 months (Heterozygous controls vs. Doubleknock-out, =8/group: 17.6±1.0 vs. 22.7±1.4 mg/dl, P=0.01). Massontrichrome staining showed an increase in glomerulosclerosis and mildinterstitial fibrosis at 12 months in the double knock-out mice comparedto heterozygous controls. However, in-depth fibrosis analysis (Totalcollagen, picrosirius red staining, Masson trichrome, and BUN) of theCtns/Vnn1 double knock-out mice did not suggest any increase in renalfibrosis with the addition of the Vnn1 gene deletion compared to Ctns−/−mice.

Example 10 Examination of Cystinosin Deficient Interstitial Macrophages

In order to examine the fibrotic response of cystinosin deficient(Ctns−/−) interstitial macrophages (Mφ) in response to renal injury(such as tubular apoptosis), a cohort of Ctns−/− mice on a C57BL/6background was followed for 12+ months.

Total renal collagen levels (μg hydroxyproline/mg) were measured in thekidneys of Ctns+/+ and Ctns−/− mice at 3 and 12 months of age. Totalcollagen in the tissue was calculated on the assumption that collagencontains 12.7% hydroxyproline by weight. Macrophage accumulation wasanalyzed by confocal staining using F4/80 rat anti-mouse macrophagemonoclonal antibody (Serotec Ltd., Oxford, UK).

As seen in FIG. 13C total kidney collagen significantly increased2.3-fold in the kidneys of Ctns−/− mice between 3 and 12 months of age.The increase in total collagen corresponded to an increase in F4/80+interstitial Mφs over the same time period. See FIGS. 13A and B.

Unilateral ureteral obstruction (UUO) was performed on 3-month oldCtns+/+ and Ctns−/− mice. Kidneys (obstructed and contralateral) werethen harvested at day 14 after UUO. Total collagen was measured ashydroxyproline concentration in hydrolysates extracted from frozennormal (contralateral) and UUO kidney samples. Macrophage accumulationin the obstructed and contralateral at day 14 was analyzed by confocalstaining using F4/80 rat anti-mouse macrophage monoclonal antibody(Serotec Ltd., Oxford, UK).

As seen in FIGS. 14A and 14B, Ctns−/− mice subjected to UUO developedsignificantly worse fibrosis (19% higher total collagen) with 63% moreF4/80+ interstitial Mφs compared to Ctns+/+ mice.

The expression pattern of cystinosin in chronically damaged kidneys wasinvestigated. UUO was performed on 8 week-old Ctns+/+C57BL/6 mice. Thekidneys (obstructed and contralateral) were then harvested at days 3, 7and 14 after UUO and semi-quantitative real-time PCR was performed withspecific primers to cystinosin gene using iCycler (Bio-Rad) using astandard protocol. Relative mRNA transcription levels of cystinosin inUUO kidneys was normalized with respect to GAPDH.

In the UUO model induced in Ctns+/+C57BL/6 mice, semi-quantitativereal-time RT-PCR revealed that Ctns gene expression initially declined(days 3 and 7), likely reflecting tubular injury, and then increased atday 14 (FIG. 15A).

RNA isolated was isolated from Ctns+/+ thioglycollate peritonealmacrophages that were either cultured alone or co-cultured withapoptotic renal tubular cells (+IRR MCT) for 24 hours. SemiquantitativeRT-PCR was performed with specific primers to cystinosin gene usingiCycler (Bio-Rad) using a standard protocol. Relative mRNA transcriptionlevels of cystinosin in the macrophages was normalized with respect toGAPDH.

Ctns mRNA expression was confirmed in wild-type peritoneal My and Ctnsexpression was increased 4-fold in macrophages after phagocytosis ofapoptotic proximal tubular cells (FIG. 15 B).

Cytokine profiling studies were performed to elucidate differences inmacrophage function between Ctns+/+ and Ctns−/− Mφ. An antibody-basedmethod of isolating kidney CD11b+ Mφ by both the AutoMACS® magnetic beadsystem and by flow cytometry (FACS) was developed and Ctns+/+ andCtns−/− CD11b+Mφ were isolated.

An in vitro model to investigate the effects of apoptotic tubular cellson macrophage activation was then developed to study the downstreameffects that are triggered by tubular apoptosis following theirphagocytic clearance. Thioglycollate peritoneal macrophages wereco-cultured with apoptotic mouse cortical tubular cells (MCT) cells for24 hours in serum free media. Cells were harvested and RNA extracted forsemiquantitative real time RT-PCR. Semi-quantitative real-time PCR wasperformed with specific primers to TNF-α, TNF-α Receptor, TGF-β, andTGF-β Receptor genes using iCycler (Bio-Rad) with a standard protocol.Relative mRNA transcription levels of TNF-α, TNF-α Receptor, TGF-β, andTGF-β Receptor in the macrophages was normalized with respect to GAPDH.

The levels of tumor necrosis factor (TNF)-α and transforming growthfactor (TGF)-β receptor mRNA were significantly higher in Ctns−/−peritoneal Mφ compared to Ctns+/+ Mφ after incubation with apoptotictubular cells for 24 hours (FIG. 16 A). Similar cytokine differenceswere observed in Ctns−/− kidneys 14 days after UUO compared to Ctns+/+kidneys (FIG. 16B).

Example 11 Plasma Cysteamine Levels in High Dose Cysteamine BitartrateTreated Mice

Since the doses of cysteamine bitartrate (400 mg/kg and 600 mg/kg)administered to mice in Examples 1-8 were much larger compared totypical doses in humans, the serum cysteamine levels of cysteaminebitartrate were measured in the mice.

Plasma cysteamine levels were measured by mass spectrometry by thereference laboratory at UC San Diego that also runs most of the humansamples for the North American patients with cystinosis. Plasma levelsof cysteamine taken at the time of sacrifice were low in the 400 mg/kgand 600 mg/kg cysteamine bitartrate treated mice at day 14 after UUO(400 mg/kg-0.81±0.09 Ilmole/L; and 600 mg/kg-1.04±0.15 Ilmole/L; levelswere undetectable in the vehicle alone group).

To test if the low levels of plasma cysteamine were attributable to theshort half life of cysteamine or the nocturnal feeding habits of themice, a more detailed analysis on the high dose cysteamine bitartrategroup (600 mg/kg) was performed. C57BL/6 mice were placed on 600 mg/kgof cysteamine bitartrate in their drinking water, changed daily, for 3days prior to sacrifice. It was assumed that an average 25 g mousedrinks 5 mLs of water per day; cysteamine bitartrate was diluted inwater and prepared daily to provide 600 mg/kg. Plasma levels were takenevery hour during the evening (for 12 hours) and every four hours duringthe day (n=4/group). Pharmacokinetic data confirms that the higher serumlevels of cysteamine are achieved at night and lower levels during theday (FIG. 17). In addition, Cmax levels in mice were found to be between15-20 μmol/L, which is similar to the levels reported in humans (Dohil Ret al., J Pediatr, 2006, 148:718-9).

Example 12 Method of Treating CKD in a Human Patient by Administrationof Cysteamine

A human patient suffering from Stage 1 CKD is identified. An effectivedose, as determined by the physician, of cysteamine is administered tothe patient. Renal function and histology is observed in the patient.Treatment is determined to be effective if minimal decrease in renalfunction is observed over a time period determined by the physician.

Example 13 Method of Treating a Human Patient at Risk for Developing CKDby Administration of Cysteamine

A human patient with a risk factor for developing CKD is identified. Thepatient is given one-quarter of a maintenance dose of cysteaminebitartrate in four divided doses administered every 6 to 8 hours. Theadministered dose is raised gradually over four to six weeks until amaintenance dose is reached. A maintenance dosage is administered to thepatient over a time period determined by the physician. Renal functionand histology is observed in the patient. Treatment is determined to beeffective if no decrease in renal function is observed over a timeperiod determined by the physician.

Example 14 Method of Treating Injury-Induced CKD in a Human Patient byAdministration of Cysteamine

A human patient with a recent kidney trauma is identified. An effectivedose, as determined by the physician, of cysteamine bitartrate isadministered to the patient. Renal function and histology is observed inthe patient. Treatment is determined to be effective if no decrease inrenal function or interstitial fibrosis is observed over a time perioddetermined by the physician.

Example 15 Method of Treating Cardiac Fibrosis in a Human Patient byAdministration of Cysteamine

A human patient with a recent cardiac infarction is identified.Cysteamine bitartrate enteric-coated is administered to the patient ineffective dose determined by the physician twice daily for a time perioddetermined by the physician. Cardiac output and histology is observed inthe patient. Treatment is determined to be effective if no decrease incardiac output is observed over a time period determined by thephysician.

Example 16 Method of Treating Liver Fibrosis in a Human Patient byAdministration of Cysteamine

A human patient suffering from liver fibrosis is identified. Cysteaminebitartrate enteric-coated is administered to the patient in effectivedose determined by the physician twice daily for a time perioddetermined by the physician. Liver function and histology is observed inthe patient. Treatment is determined to be effective if cirrhosis of theliver does not develop during a time period determined by the physician.

Example 17 Method of Treating a Human Hepatitus Patient byAdministration of Cysteamine

A human patient suffering from hepatitis is identified. An effectivedose, as determined by the physician, of cysteamine is administered tothe patient for a period of time determined by the physician. Liverfunction and histology is observed in the patient. Treatment isdetermined to be effective if cirrhosis of the liver does not developduring a time period determined by the physician.

Example 18 Method of Treating Interstitial Lung Disease in a HumanPatient by Administration of Cysteamine

A human patient suffering from interstitial lung disease is identified.An effective dose, as determined by the physician, of cysteamine isadministered to the patient for a period of time determined by thephysician. Respiratory function and histology is observed in thepatient. Treatment is determined to be effective if an increase inrespiratory function is observed over a time period determined by thephysician.

Example 19 Method of Treating CKD in a Human Patient by Administrationof Cystamine

A human patient suffering from Stage 1 CKD is identified. An effectivedose, as determined by the physician, of cystamine is administered tothe patient. Renal function and histology is observed in the patient.Treatment is determined to be effective if minimal decrease in renalfunction is observed over a time period determined by the physician.

Example 20 Method of Treating a Human Patient at Risk for Developing CKDby Administration of Cystamine

A human patient with a risk factor for developing CKD is identified. Thepatient is given one-quarter of a maintenance dose of cystamine in fourdivided doses administered every 6 to 8 hours. The administered dose israised gradually over four to six weeks until a maintenance dose isreached. A maintenance dosage is administered to the patient over a timeperiod determined by the physician. Renal function and histology isobserved in the patient. Treatment is determined to be effective if nodecrease in renal function is observed over a time period determined bythe physician.

Example 21 Method of Treating Injury-Induced CKD in a Human Patient byAdministration of Cystamine

A human patient with a recent kidney trauma is identified. An effectivedose, as determined by the physician, of cystamine is administered tothe patient. Renal function and histology is observed in the patient.Treatment is determined to be effective if no decrease in renal functionor interstitial fibrosis is observed over a time period determined bythe physician.

1. A method of treating a fibrotic disease comprising administering, toa patient in need thereof, an effective amount of cysteamine, or a saltthereof; wherein the administration of cysteamine, or a salt thereof,results in amelioration of a fibrotic disease in the patient. 2-22.(canceled)
 23. A method for treating a disorder associated with elevatedlevels of interstitial extracellular matrix (ECM) in a tissue, saidmethod comprising administering, to a patient diagnosed with thedisorder, an effective amount of cysteamine, or a salt thereof; whereinthe administration of cysteamine, or a salt thereof, results inreduction or maintenance of the level of interstitial ECM in the tissueof the patient.
 24. The method, according to claim 1, wherein the saltof cysteamine is cysteamine bitartrate.
 25. The method of claim 1,further comprising administering a TGF-β antagonist to said patient. 26.The method of claim 1, further comprising administering a TNF-αantagonist to said patient.
 27. The method of claim 1, wherein theeffective amount of cysteamine, or salt thereof, is from 1 mg to 40 mgper kilogram of body weight daily.
 28. The method of claim 27, whereinthe effective amount of cysteamine, or salt thereof, is from 15 mg to 20mg per kilogram of body weight.
 29. The method of claim 1, wherein thefibrotic disease is selected from the group consisting of:atherosclerosis, asthma, cardiac fibrosis, organ transplant fibrosis,colloid and hypertrophic scar, muscle fibrosis, pancreatic fibrosis,bone-marrow fibrosis, interstitial liver fibrosis, cirrhosis of liverand gallbladder, scleroderma, pulmonary fibrosis, diffuse parenchymallung disease, idiopathic interstitial fibrosis, interstitialpneumonitis, desquamative interstitial pneumonia, respiratorybronchiolitis, interstitial lung disease, acute interstitialpneumonitis, nonspecific interstitial pneumonia, cryptogenic organizingpneumonia, lymphocytic interstitial pneumonia, renal fibrosis, andchronic kidney disease.
 30. The method of claim 23, wherein the tissuecomprises an organ selected from the group consisting of lung, heart,blood vessel, liver, gallbladder, kidney, skin, lung, muscle, pancreas,and thyroid.
 31. A method for reducing the progression of chronic kidneydisease (CKD), said method comprising administering, to a patientdiagnosed with CKD, an effective amount of cysteamine, or a saltthereof; wherein the administration of cysteamine, or a salt thereof,results in reducing CKD progression in the patient.
 32. The method,according to claim 31, wherein the salt of cysteamine is cysteaminebitartrate.
 33. The method of claim 31, wherein the effective amount ofcysteamine, or salt thereof, is about 1 mg to about 3 g daily.
 34. Themethod of claim 33, wherein the effective amount of cysteamine, or saltthereof, is about 10 mg to about 2 g daily.
 35. The method of claim 31,wherein the patient is diagnosed with Stage 1, 2, 3, or 4 CKD.
 36. Themethod of claim 1, wherein the disease is chronic kidney disease and themethod results in reduction of interstitial fibrosis in the patient 37.The method of claim 36, wherein the reduction of interstitial fibrosisis measured by a decrease in ECM accumulation
 38. The method of claim36, wherein the patient is diagnosed with diabetes mellitus or hassuffered kidney trauma.
 39. The method, according to claim 36, whereinthe salt of cysteamine is cysteamine bitartrate.
 40. The method of claim36, wherein the effective amount of cysteamine, or salt thereof, is from1 grams/m²/day to about 3 grams/m²/day.
 41. The method of claim 40,wherein the effective amount of cysteamine, or salt thereof, is from1.30 grams/m²/day to 1.95 grams/m²/day.
 42. A method of amelioratingfibrosis in a patient, comprising: identifying a patient with fibrosis;and providing to said patient cysteamine, or a salt thereof, wherein anamelioration of fibrosis in said patient is observed.
 43. The method ofclaim 36, wherein the patient is at risk for developing chronic kidneydisease.